Bispecific antibodies specific for OX40

ABSTRACT

The invention relates to antibody or antibody fragments thereof that bind to OX40, methods of producing these antibodies and fragments, and to methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119 toEuropean Patent Application No. 15188095.2, filed Oct. 2, 2015 andEuropean Patent Application No. 16170363.2, filed May 19, 2016, whichapplications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and is hereby incorporated by reference in its entirety. SaidASCII copy, created on Sep. 13, 2016, is named P33116US_SeqList.txt, andis 698,591 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel bispecific antigen binding molecules,comprising (a) at least one moiety capable of specific binding to acostimulatory TNF receptor family member, (b) at least one moietycapable of specific binding to a target cell antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation. The invention further relates to methods of producing thesemolecules and to methods of using the same.

BACKGROUND

Several members of the tumor necrosis factor receptor (TNFR) familyfunction after initial T cell activation to sustain T cell responses andthus have pivotal roles in the organization and function of the immunesystem. CD27, 4-1BB (CD137), OX40 (CD134), HVEM, CD30, and GITR can havecostimulatory effects on T cells, meaning that they sustain T-cellresponses after initial T cell activation (Watts T. H. (2005) Annu. Rev.Immunol. 23, 23-68). The effects of these costimulatory TNFR familymembers can often be functionally, temporally, or spatially segregatedfrom those of CD28 and from each other. The sequential and transientregulation of T cell activation/survival signals by differentcostimulators may function to allow longevity of the response whilemaintaining tight control of T cell survival. Depending on the diseasecondition, stimulation via costimulatory TNF family members canexacerbate or ameliorate disease. Despite these complexities,stimulation or blockade of TNFR family costimulators shows promise forseveral therapeutic applications, including cancer, infectious disease,transplantation, and autoimmunity.

Among several costimulatory molecules, the tumor necrosis factor (TNF)receptor family member OX40 (CD134) plays a key role in the survival andhomeostasis of effector and memory T cells (Croft M. et al. (2009),Immunological Reviews 229, 173-191). OX40 (CD134) is expressed inseveral types of cells and regulates immune responses againstinfections, tumors and self-antigens and its expression has beendemonstrated on the surface of T-cells, NKT-cells and NK-cells as wellas neutrophils (Baumann R. et al. (2004), Eur. J. Immunol. 34,2268-2275) and shown to be strictly inducible or strongly upregulated inresponse to various stimulatory signals. Functional activity of themolecule has been demonstrated in every OX40-expressing cell typesuggesting complex regulation of OX40-mediated activity in vivo.Combined with T-cell receptor triggering, OX40 engagement on T-cells byits natural ligand or agonistic antibodies leads to synergisticactivation of the PI3K and NFκB signalling pathways (Song J. et al.(2008) J. Immunology 180(11), 7240-7248). In turn, this results inenhanced proliferation, increased cytokine receptor and cytokineproduction and better survival of activated T-cells. In addition to itsco-stimulatory activity in effector CD4⁺ or CD8⁺ T-cells, OX40triggering has been recently shown to inhibit the development andimmunosuppressive function of T regulatory cells. This effect is likelyto be responsible, at least in part, for the enhancing activity of OX40on anti-tumor or anti-microbial immune responses. Given that OX40engagement can expand T-cell populations, promote cytokine secretion,and support T-cell memory, agonists including antibodies and solubleforms of the ligand OX40L have been used successfully in a variety ofpreclinical tumor models (Weinberg et al. (2000), J. Immunol. 164,2160-2169).

4-1BB (CD137), a member of the TNF receptor superfamily, has been firstidentified as a molecule whose expression is induced by T-cellactivation (Kwon Y. H. and Weissman S. M. (1989), Proc. Natl. Acad. Sci.USA 86, 1963-1967). Subsequent studies demonstrated expression of 4-1BBin T- and B-lymphocytes (Snell L. M. et al. (2011) Immunol. Rev. 244,197-217 or Zhang X. et al. (2010), J. Immunol. 184, 787-795), NK-cells(Lin W. et al. (2008), Blood 112, 699-707, NKT-cells (Kim D. H. et al.(2008), J. Immunol. 180, 2062-2068), monocytes (Kienzle G. and vonKempis J. (2000), Int. Immunol. 12, 73-82, or Schwarz H. et al. (1995),Blood 85, 1043-1052), neutrophils (Heinisch I. V. et al. (2000), Eur. J.Immunol. 30, 3441-3446), mast (Nishimoto H. et al. (2005), Blood 106,4241-4248), and dendritic cells as well as cells of non-hematopoieticorigin such as endothelial and smooth muscle cells (Broll K. et al.(2001), Am. J. Clin. Pathol. 115, 543-549 or Olofsson P. S. et al.(2008), Circulation 117, 1292-1301). Expression of 4-1BB in differentcell types is mostly inducible and driven by various stimulatorysignals, such as T-cell receptor (TCR) or B-cell receptor triggering, aswell as signaling induced through co-stimulatory molecules or receptorsof pro-inflammatory cytokines (Diehl L. et al. (2002), J. Immunol. 168,3755-3762; von Kempis J. et al. (1997), Osteoarthritis Cartilage 5,394-406; Zhang X. et al. (2010), J. Immunol. 184, 787-795).

CD137 signaling is known to stimulate IFNγ secretion and proliferationof NK cells (Buechele C. et al. (2012), Eur. J. Immunol. 42, 737-748;Lin W. et al. (2008), Blood 112, 699-707; Melero I. et al. (1998), CellImmunol. 190, 167-172) as well as to promote DC activation as indicatedby their increased survival and capacity to secret cytokines andupregulate co-stimulatory molecules (Choi B. K. et al. (2009), J.Immunol. 182, 4107-4115; Futagawa T. et al. (2002), Int. Immunol. 14,275-286; Wilcox R. A. et al. (2002), J. Immunol. 168, 4262-4267).However, CD137 is best characterized as a co-stimulatory molecule whichmodulates TCR-induced activation in both the CD4⁺ and CD8⁺ subsets ofT-cells. In combination with TCR triggering, agonistic 4-1BB-specificantibodies enhance proliferation of T-cells, stimulate lymphokinesecretion and decrease sensitivity of T-lymphocytes toactivation-induced cells death (Snell L. M. et al. (2011) Immunol. Rev.244, 197-217). In line with these co-stimulatory effects of 4-1BBantibodies on T-cells in vitro, their administration to tumor bearingmice leads to potent anti-tumor effects in many experimental tumormodels (Melero I. et al. (1997), Nat. Med. 3, 682-685; Narazaki H. etal. (2010), Blood 115, 1941-1948). In vivo depletion experimentsdemonstrated that CD8⁺ T-cells play the most critical role inanti-tumoral effect of 4-1BB-specific antibodies. However, depending onthe tumor model or combination therapy, which includes anti-4-1BB,contributions of other types of cells such as DCs, NK-cells or CD4⁺T-cells have been reported (Murillo O. et al. (2009), Eur. J. Immunol.39, 2424-2436; Stagg J. et al. (2011), Proc. Natl. Acad. Sci. USA 108,7142-7147).

In addition to their direct effects on different lymphocyte subsets,4-1BB agonists can also induce infiltration and retention of activatedT-cells in the tumor through 4-1BB-mediated upregulation ofintercellular adhesion molecule 1 (ICAM1) and vascular cell adhesionmolecule 1 (VCAM1) on tumor vascular endothelium (Palazon A. et al.(2011), Cancer Res. 71, 801-811). 4-1BB triggering may also reverse thestate of T-cell anergy induced by exposure to soluble antigen that maycontribute to disruption of immunological tolerance in the tumormicro-environment or during chronic infections (Wilcox R. A. et al.(2004), Blood 103, 177-184).

It appears that the immunomodulatory properties of 4-1BB agonisticantibodies in vivo require the presence of the wild type Fc-portion onthe antibody molecule thereby implicating Fc-receptor binding as animportant event required for the pharmacological activity of suchreagents as has been described for agonistic antibodies specific toother apoptosis-inducing or immunomodulatory members of theTNFR-superfamily (Li F. and Ravetch J. V. (2011), Science 333,1030-1034; Teng M. W. et al. (2009), J. Immunol. 183, 1911-1920).However, systemic administration of 4-1BB-specific agonistic antibodieswith the functionally active Fc domain also induces expansion of CD8⁺T-cells associated with liver toxicity (Dubrot J. et al. (2010), CancerImmunol. Immunother. 59, 1223-1233) that is diminished or significantlyameliorated in the absence of functional Fc-receptors in mice. In humanclinical trials (ClinicalTrials.gov, NCT00309023), Fc-competent 4-1BBagonistic antibodies (BMS-663513) administered once every three weeksfor 12 weeks induced stabilization of the disease in patients withmelanoma, ovarian or renal cell carcinoma. However, the same antibodygiven in another trial (NCT00612664) caused grade 4 hepatitis leading totermination of the trial (Simeone E. and Ascierto P. A. (2012), J.Immunotoxicology 9, 241-247). Thus, there is a need for new generationagonists that should not only effectively engage 4-1BB on the surface ofhematopoietic and endothelial cells but also be capable of achievingthat through mechanisms other than binding to Fc-receptors in order toavoid uncontrollable side effects.

The available pre-clinical and clinical data clearly demonstrate thatthere is a high clinical need for effective agonists of costimulatoryTNFR family members such as Ox40 and 4-1BB that are able to induce andenhance effective endogenous immune responses to cancer. However, almostnever are the effects limited to a single cell type or acting via asingle mechanism and studies designed to elucidate inter- andintracellular signaling mechanisms have revealed increasing levels ofcomplexity. Thus, there is a need of “targeted” agonists that preferablyact on a single cell type. The antigen binding molecules of theinvention combine a moiety capable of preferred binding totumor-specific or tumor-associated targets with a moiety capable ofagonistic binding to costimulatory TNF receptors. The antigen bindingmolecules of this invention may be able to trigger TNF receptors notonly effectively, but also very selectively at the desired site therebyreducing undesirable side effects.

SUMMARY OF THE INVENTION

The present invention relates to bispecific antigen binding moleculescombining at least one moiety capable of specific binding to acostimulatory TNF receptor family member, i.e at least one antigenbinding site that targets costimulatory TNF receptors with at least onemoiety capable of specific binding to a target cell antigen, i.e. withat least one antigen binding side targeting a target cell antigen. Thesebispecific antigen binding molecules are advantageous as they willpreferably activate costimulatory TNF receptors at the site where thetarget cell antigen is expressed, due to their binding capabilitytowards a target cell antigen. The invention also provides novelantibodies capable of specific binding to a costimulatory TNF receptorfamily member. In comparison to known antibodies to costimulatory TNFreceptors these antibodies have properties that are advantageous forincorporating them into bispecific antigen binding molecules incombination with moieties capable of specific binding to a target cellantigen.

In one aspect, the invention provides a bispecific antigen bindingmolecule, comprising

-   (a) at least one moiety capable of specific binding to a    costimulatory TNF receptor family member,-   (b) at least one moiety capable of specific binding to a target cell    antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In a particular aspect, the bispecific antigen binding moleculecomprises (a) at least one moiety capable of specific binding to acostimulatory TNF receptor family member, wherein the costimulatory TNFreceptor family member is selected from the group consisting of OX40 and4-1BB, (b) at least one moiety capable of specific binding to a targetcell antigen, and (c) a Fc domain composed of a first and a secondsubunit capable of stable association.

In one aspect, the costimulatory TNF receptor family member is OX40.Thus, in a particular aspect, the moiety capable of specific binding toa costimulatory TNF receptor family member binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:1.

In a further aspect, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to OX40,wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:2 and SEQ ID NO:3,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:4 and SEQ ID NO:5, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,        SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID        NO:15,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID        NO:18, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

In another aspect, the invention provides a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to OX40comprises a heavy chain variable region VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35 and SEQ ID NO:37 and a light chain variable regionVL comprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ IDNO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36 and SEQ ID NO:38.

Particularly, a bispecific antigen binding molecule is provided, whereinthe moiety capable of specific binding to OX40 comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

In another aspect, the costimulatory TNF receptor family member is4-1BB. Thus, in a particular aspect, the moiety capable of specificbinding to a costimulatory TNF receptor family member binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:39.

Furthermore, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to 4-1BB,wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:40 and SEQ ID NO:41,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:42 and SEQ ID NO:43, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:44, SEQ ID NO:45, SEQ ID        NO:46, SEQ ID NO:47 and SEQ ID NO:48        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:49 and SEQ ID NO:50,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:51 and SEQ ID NO:52, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ ID        NO:55, SEQ ID NO:56 and SEQ ID NO:57.

In another aspect, the invention provides a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to 4-1BBcomprises a heavy chain variable region VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 and SEQ IDNO:66 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 and SEQ IDNO:67.

Particularly, a bispecific antigen binding molecule is provided, whereinthe moiety capable of specific binding to 4-1BB comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

In a particular aspect, the invention provides a bispecific antigenbinding molecule, comprising

-   (a) at least one moiety capable of specific binding to a    costimulatory TNF receptor family member,-   (b) at least one moiety capable of specific binding to a target cell    antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association, wherein the moiety capable of specific binding    to a costimulatory TNF receptor family member is a Fab fragment. In    one aspect, if the bispecific antigen binding molecule, comprises    more than one moiety capable of specific binding to a costimulatory    TNF receptor family member, all moieties capable of specific binding    to a costimulatory TNF receptor family member are Fab fragments.

In another aspect, the bispecific antigen binding molecule comprises (a)at least one moiety capable of specific binding to a costimulatory TNFreceptor family member, (b) at least one moiety capable of specificbinding to a target cell antigen, wherein the target cell antigen isselected from the group consisting of Fibroblast Activation Protein(FAP), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA),CD19, CD20 and CD33, and (c) a Fc domain composed of a first and asecond subunit capable of stable association. More particularly, thetarget cell antigen is Fibroblast Activation Protein (FAP).

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to FAPcomprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:68 and SEQ ID NO:69,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:70 and SEQ ID NO:71, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:72 and SEQ ID NO:73,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:74 and SEQ ID NO:75,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:76 and SEQ ID NO:77, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:78 and SEQ ID NO:79.

In a further aspect, the invention thus provides a bispecific antigenbinding molecule, wherein

-   (i) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29,    SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID    NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

In one aspect, the invention provides a bispecific antigen bindingmolecule, wherein

-   (i) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising the amino acid sequence of SEQ    ID NO: 27 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO: 28 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:82 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:83. In another aspect, the invention provides    a bispecific antigen binding molecule, wherein-   (i) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,    SEQ ID NO:64 or SEQ ID NO:66 and a light chain variable region    comprising an amino acid sequence that is at least about 95%, 96%,    97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID    NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

In a further aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to a        costimulatory TNF receptor family member,    -   (b) a second Fab fragment capable of specific binding to a        target cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In one aspect, the bispecific antigen binding molecule is a human, ahumanized or a chimeric antibody. In particular, the Fc domain is ahuman IgG domain, particularly an IgG1 Fc domain or an IgG4 Fc domain.

In a further aspect, provided is a bispecific antigen binding molecule,wherein the Fc domain comprises one or more amino acid substitution thatreduces the binding affinity of the antibody to an Fc receptor and/oreffector function. In particular, the Fc domain is of human IgG1subclass with the amino acid mutations L234A, L235A and P329G (numberingaccording to Kabat EU index).

In a further aspect, provided is a bispecific antigen binding molecule,wherein the Fc domain comprises a modification promoting the associationof the first and second subunit of the Fc domain. In a particularaspect, the invention provides a bispecific antigen binding molecule,wherein the first subunit of the Fc domain comprises knobs and thesecond subunit of the Fc domain comprises holes according to the knobsinto holes method. More particularly, the first subunit of the Fc domaincomprises the amino acid substitutions S354C and T366W (numberingaccording to Kabat EU index) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

In particular the invention provides a bispecific antigen bindingmolecule, comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) two moieties capable of specific binding to a target cell    antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is bivalent both for thecostimulatory TNF receptor family member and for the target cellantigen.

In a particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) two additional Fab fragments capable of specific binding to a    target cell antigen, wherein said additional Fab fragments are each    connected via a peptide linker to the C-terminus of the heavy chains    of (a). More particularly, the two additional Fab fragments capable    of specific binding to a target cell antigen are cross-Fab fragments    wherein the variable domains VL and VH are replaced by each other    and the VL-CH chains are each connected via a peptide linker to the    C-terminus of the heavy chains of (a).

In one aspect, the two Fab fragments capable of specific binding to acostimulatory TNF receptor family member are two Fab fragments capableof specific binding to OX40 or 4-1BB and the two additional Fabfragments capable of specific binding to a target cell antigen arecross-Fab fragments capable of specific binding to FAP.

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) one moiety capable of specific binding to a target cell antigen,    and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is bivalent for the costimulatoryTNF receptor family member and monovalent for the target cell antigen.

In a particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of one of the heavy chains and wherein the VL domain    is connected via a peptide linker to the C-terminus of the second    heavy chain.

In another aspect, the invention provides an antibody that specificallybinds to OX40, wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

In yet another aspect, provided is an antibody that specifically bindsto 4-1BB, wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

According to another aspect of the invention, there is provided anisolated polynucleotide encoding a bispecific antigen binding moleculeas described herein before or an antibody that specifically binds toOX40 as described herein before or an antibody that specifically bindsto 4-1BB as described herein before. The invention further provides avector, particularly an expression vector, comprising the isolatedpolynucleotide of the invention and a host cell comprising the isolatedpolynucleotide or the vector of the invention. In some aspects the hostcell is a eukaryotic cell, particularly a mammalian cell.

In another aspect, provided is a method for producing a bispecificantigen binding molecule as described herein before or an antibody thatspecifically binds to OX40 as described herein before or an antibodythat specifically binds to 4-1BB as described herein before, comprisingthe steps of (i) culturing the host cell of the invention underconditions suitable for expression of the antigen binding molecule, and(ii) recovering the antigen binding molecule. The invention alsoencompasses the bispecific antigen binding molecule or the antibody thatspecifically binds to OX40 or the antibody that specifically binds to4-1BB produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising abispecific antigen binding molecule as described herein before or anantibody that specifically binds to OX40 as described herein before oran antibody that specifically binds to 4-1BB as described herein beforeand at least one pharmaceutically acceptable excipient.

Also encompassed by the invention is the bispecific antigen bindingmolecule as described herein before or the antibody that specificallybinds to OX40 as described herein before or the antibody thatspecifically binds to 4-1BB as described herein before, or thepharmaceutical composition comprising the bispecific antigen bindingmolecule or the antibody that specifically binds to OX40 or the antibodythat specifically binds to 4-1BB, for use as a medicament.

In one aspect, provided is a bispecific antigen binding molecule asdescribed herein before or an antibody that specifically binds to OX40as described herein before or an antibody that specifically binds to4-1BB as described herein before or the pharmaceutical composition ofthe invention, for use

-   (i) in stimulating T cell response,-   (ii) in supporting survival of activated T cells,-   (iii) in the treatment of infections,-   (iv) in the treatment of cancer,-   (v) in delaying progression of cancer, or-   (vi) in prolonging the survival of a patient suffering from cancer.

In a specific embodiment, provided is the bispecific antigen bindingmolecule as described herein before or the antibody that specificallybinds to OX40 as described herein before or the antibody thatspecifically binds to 4-1BB as described herein before, or thepharmaceutical composition of the invention, for use in the treatment ofcancer.

In another specific aspect, the invention provides the bispecificantigen binding molecule as described herein before or the antibody thatspecifically binds to OX40 as described herein before or the antibodythat specifically binds to 4-1BB as described herein for use in thetreatment of cancer, wherein the bispecific antigen binding molecule isadministered in combination with a chemotherapeutic agent, radiationand/or other agents for use in cancer immunotherapy.

In a further aspect, the invention provides a method of inhibiting thegrowth of tumor cells in an individual comprising administering to theindividual an effective amount of the bispecific antigen bindingmolecule as described herein before or the antibody that specificallybinds to OX40 as described herein before or the antibody thatspecifically binds to 4-1BB as described herein before, or thepharmaceutical composition of the invention, to inhibit the growth ofthe tumor cells.

Also provided is the use of the bispecific antigen binding molecule asdescribed herein before or the antibody that specifically binds to OX40as described herein before or the antibody that specifically binds to4-1BB as described herein before for the manufacture of a medicament forthe treatment of a disease in an individual in need thereof, inparticular for the manufacture of a medicament for the treatment ofcancer, as well as a method of treating a disease in an individual,comprising administering to said individual a therapeutically effectiveamount of a composition comprising the TNF family ligandtrimer-containing antigen binding molecule of the invention in apharmaceutically acceptable form. In a specific aspect, the disease iscancer. In any of the above aspects the individual is a mammal,particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the monomeric form of Fc-linked TNF receptor antigen thatwas used for the preparation of TNF receptor antibodies.

FIG. 1B shows a dimeric human TNF receptor antigen Fc fusion moleculewith a C-terminal Ha tag that was used for the testing of the binding ofTNF receptor antibodies in the presence of TNF ligand (ligand blockingproperty).

FIG. 1C shows the schematic setup of the experiment described in Example2.

FIGS. 2A, 2B, 2C and 2D show the binding of anti-OX40 antibodies toactivated human CD4⁺ and CD8⁺ T cells. OX40 is not expressed on restinghuman PBMCs (FIGS. 2A and 2C). After activation of human PBMCs OX40 isup-regulated on CD4⁺ and CD8⁺ T cells (FIGS. 2B and 2D). OX40 expressionon human CD8⁺ T cells is lower than on CD4⁺ T cells. The depicted clonesvaried in their binding strength (EC₅₀ values as well as signalstrength) to OX40 positive cells. Shown is the binding as median offluorescence intensity (MFI) of FITC labeled anti-human IgG Fcγ-specificgoat IgG F(ab′)2 fragment which is used as secondary detection antibody.MFI was measured by flow cytometry and baseline corrected by subtractingthe MFI of the blank control. The x-axis shows the concentration ofantibody constructs. All OX40 clones do bind to activated, OX40expressing human CD4⁺ T cells, and to a lower extent to activated humanCD8⁺ T cells.

FIGS. 3A, 3B, 3C and 3D show the binding of the anti-OX40 antibodies toactivated mouse CD4⁺ and CD8⁺ T cells. OX40 was not detected on restingmouse splencoytes (FIGS. 3A and 3C). After activation OX40 isup-regulated on CD4⁺ and CD8⁺ T cells (FIGS. 3B and 3D). Mousesplenocytes were isolated by erythrolysis with ACK lysis buffer ofmechanically-homogenized spleens obtained from 6-8 weeks old femaleC57BL/6 mice. Binding of anti-OX40 antibodies to cell surface proteinswas detected with a goat anti-human IgG Fc-specific secondary antibodyconjugated to FITC using FACS analysis. MFI was measured by flowcytometry and baseline corrected by subtracting the MFI of the blankcontrol. The x-axis shows the concentration of antibody constructs. Onlyclone 20B7 does bind to activated, OX40 expressing mouse CD4 and CD8 TCells, but not to resting T cells.

FIGS. 4A and 4B show the binding of anti-OX40 antibodies on cynomolgusactivated CD4⁺ and CD8⁺ T cells. The depicted clones varied in theirbinding strength (EC₅₀ values as well as signal strength) to OX40positive activated cynomolgus CD4⁺ T cells (FIG. 4A). OX40 expression onactivated CD8⁺ T cells is low under this condition and hardly anybinding of the selected clones was found (FIG. 4B). Binding of anti-OX40antibodies to cell surface proteins was detected with a goat anti-humanIgG Fe-specific secondary antibody conjugated to FITC using FACSanalysis. MFI was measured by flow cytometry and baseline corrected bysubtracting the MFI of the blank control. The x-axis shows theconcentration of antibody constructs. All OX40 clones do bind toactivated, OX40 expressing cynomolgus CD4⁺ T cells, and to a lowerextent to activated cynomolgus CD8⁺ T cells.

FIGS. 5A and 5B show the lack of binding to OX40 negative tumor cells.The depicted clones showed no binding to OX40 negative U-78 MG (FIG. 5A)and WM266-4 tumor cells (FIG. 5B). Shown is the binding as median offluorescence intensity (MFI) of FITC labeled anti-human IgG Fcγ-specificgoat IgG F(ab′)2 fragment which is used as secondary detection antibody.MFI was measured by flow cytometry and baseline corrected by subtractingthe MFI of the blank control. The x-axis shows the concentration ofantibody constructs. All clones in an IgG format do not bind to OX40negative tumor cells. Binding is specific for OX40 on activatedleukocytes.

FIGS. 6A, 6B, 6C, 6D, 6E and 6F show the interaction between anti-Ox40antibodies 8H9 (FIG. 6A), 20B7 (FIG. 6B), 1G4 (FIG. 6C), 49B4 (FIG. 6D),CLC-563 (FIG. 6E) and CLC-564 (FIG. 6F) and the preformed complex huOx40Ligand/huOx40-Fc as measured by surface plasmon resonance.

FIGS. 7A and 7B show the effect of the anti-human OX40 antibodies of theinvention on HeLa cells expressing human OX40 and reporter geneNF-κB-luciferase. Shown is the activation of NF-κB signaling pathway inthe reporter cell line with various anti-OX40 binders in a P329GLALAhuIgG1 format with (FIG. 7B) or without (FIG. 7B) crosslinking bysecondary-antibody. The reporter cells were cultured for 6 hours in thepresence of anti-OX40 constructs at the indicated concentrations w/ orw/o crosslinking secondary poly-clonal anti-huIgG1 Fcγ-specific goat IgGF(ab)2 fragment in a 1:2 ratio. Activity is characterized by blottingthe units of released light (URL) measured during 0.5 s versus theconcentration in nM of tested anti-Ox40 construct. URLs are emitted dueto luciferase-mediated oxidation of luciferin to oxyluciferin. Allclones are able to induce NFκB activation when the OX40 axis istriggered in a human OX40⁺ reporter cell line. All clones are thusagonistic and activate in a dose dependent way. Crosslinking bysecondary Fc part specific Abs strongly increases this agonism.

FIGS. 8A, 8B, 8C, 8D, 8E and 8F show the bioactivity of the anti-humanOX40 antibodies in preactivated human CD4 T cells. Costimulation withplate-immobilized anti-Ox40 binders (huIgG1 P329GLALA format) promotedcell proliferation and maturation of sub-optimally restimulated humanCD4 T cells and induced an enhanced activated phenotype. PHA-Lpre-activated CFSE-labeled human CD4 T cells were cultured for four dayson plates pre-coated with mouse IgG Fcγ specific antibodies, human IgGFcγ specific antibodies (both 2 μg/mL), mouse anti-human CD3 antibodies(clone OKT3, [3 ng/mL]) and titrated anti-Ox40 binders (huIgG1 P329GLALAformat). Shown is the event count (FIG. 8A), the percentage ofproliferating (CFSE-low) cells (FIG. 8B), the percentage of effector Tcells (CD127 low/CD45RA low) (FIG. 8C) and the percentage of CD62L low(FIG. 8D), OX40 positive (FIG. 8F) or Tim-3 positive cells (FIG. 8E) atday 4. Baseline values of samples containing only the plate-immobilizedanti-human CD3 were substracted. Therefore, the enhancing effect of OX40stimulation but not the effect of suboptimal anti-CD3 stimulation per seis visible here. All clones are able support suboptimal TCR stimulationin OX40 positive preactived CD4 T cells when they are coated to plate.Cells do survive better and proliferate more. In the tumormicroenvironment this could lead to increased anti-tumor activity of Tcells.

FIG. 9 summarizes the EC₅₀ values (for all biomarkers) as marker for theagonistic capacity of the respective clone (values calculated from thecurves shown in FIG. 8). The potency increases from left to right. Theevent count, the percentage of proliferating (CFSE-low) cells and thepercentage of CD62L low, CD45RA low or Tim-3 positive cells at day 4were plotted vs the anti-Ox40 antibody concentration and EC₅₀ valueswere calculated using the inbuilt sigmoidal dose response quotation inPrism4 (GraphPad Software, USA).

FIGS. 10A, 10B, 10C, 10D, 10E and 10F show the bioactivity of theanti-human OX40 antibodies in preactivated human CD4 T cells insolution. No effect on cell proliferation, maturation or activationstatus of sub-optimally restimulated human CD4 T cells was detected inthe absence of plate immobilization of anti-Ox40 binders (hu IgG1P329GLALA format). PHA-L pre-activated CFSE-labeled human CD4 T cellswere cultured for four days on plates pre-coated with mouse IgG Fcγspecific antibodies and mouse anti-human CD3 antibodies (clone OKT3, [3ng/mL]). Titrated anti-Ox40 binders (hu IgG1 P329GLALA format) wereadded to the media and were present in solution throughout theexperiment. Shown is the event count (FIG. 10A), the percentage ofproliferating (CFSE-low) cells (FIG. 10B), the percentage of effector Tcells (CD127low CD45RAlow) (FIG. 10C) and the percentage of CD62L low(FIG. 10E), OX40 positive (FIG. 10F) or Tim-3 positive cells (FIG. 10E)at day 4. Baseline values of samples containing only theplate-immobilized anti-human CD3 were substracted. Therefore, theenhancing effect of OX40 stimulation but not the effect of suboptimalanti-CD3 stimulation per se is visible here. There is no improved TCRstimulation in the absence of strong crosslinking (P329GLALA format insolution). Crosslinking is therefore essential for a bivalent aOx40format to be agonistic on T cells. This crosslinking will be provided byFAP expressed on the cell surface of tumor or tumor-stromal cells intargeted formats.

FIG. 11 shows a correlation between the binding strength and agonisticcapacity of the different anti-OX40 clones. Binding of anti-Ox40 clones(huIgG1 P329GLALA format) on activated CD4 T cells was performed asdescribed in Example 2.1.2. Plateau values were normalized to the valueobtained with clone 8H9 (huIgG1 P329GLALA format). Bioactivity testingof anti-Ox40 clones (huIgG1 P329GLALA format) was performed as describedin Example 3.2 and plateau values of PD-1 expression were normalized tothe values obtained for clone 8H9 (huIgG1 P329GLALA format). Normalizedbinding was plotted against normalized bioactivity, to test for acorrelation between binding strength and agonistic capacity. For mostclones there was a direct correlation (linear regression is shown, pvalue 0.96; slope 0.91). However, two clones (49B4, 1G4) showed a muchstronger bioactivity then could be predicted from their bindingstrength. This subgroup of clones which show unexpectedly high agonisticpotency in the face of low binding ability is of particular interest forthe bispecific antigen binding molecules of the invention.

FIG. 12A shows a schematic scheme of an exemplary bispecific, bivalentantigen binding molecule of the invention comprising two Fab fragmentsbinding to OX40 and two cross-Fab fragments binding to FAP (2+2 format).

FIG. 12B shows a schematic scheme of an exemplary bispecific, monovalentantigen binding molecule (1+1 format) of the invention comprising oneFab fragment binding to OX40 and one cross-Fab fragment binding to FAP.

FIG. 12C shows the setup for the SPR experiments showing simultaneousbinding to immobilized human OX40 or human 4-1BB and human FAP.

FIG. 12D shows a schematic drawing of an exemplary bispecific antigenbinding molecule, that is bivalent for binding to OX40 and monovalentfor binding to FAP. It comprises two Fab fragments binding to OX40 and aVH and VL domain binding to FAP. The black point symbolizes the knobinto hole modifications in the heavy chains.

FIG. 12E shows the setup for the SPR experiments demonstrating bindingto FAP as described in Example 5.4.1.

FIG. 12F shows how simultaneous binding to immobilized human OX40 andhuman FAP was measured (Example 5.4.1).

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H and 13J, 13K, 13L and 13Mshow the SPR diagrams of simultaneous binding of bispecific bivalent 2+2constructs (analyte 1) to immobilized human OX40 and human FAP (analyte2). In FIGS. 13E, 13F, 13G and 13H, the simultaneous binding ofbispecific monovalent 1+1 constructs (analyte 1) to immobilized humanOX40 and human FAP (analyte 2) is shown. In FIGS. 13J, 13K and 13L thesimultaneous binding of bispecific 2+1 constructs (analyte 1) toimmobilized human OX40 and human FAP (analyte 2) is shown. FIG. 13Mshows the binding to hu OX40 of the bispecific 2+1 constructs in acell-based FRET assay (TagLite) (Example 5.4.2). The data show that thetwo anti-OX40 Fab domains in the 2+1 constucts bind to hu OX40 incomparable manner as a common IgG antibody.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G and 14H show the binding ofselected anti-OX40 binders (clone 8H9, 1G4) in a FAP targeted monovalentor bivalent format to resting and activated human PBMC, respectively.Binding characteristics to OX40 positive T cells (FIGS. 14B and 14D)were comparable for clones in a conventional bivalent hu IgG format(open square) and a FAP-targeted bivalent format (filled square).Binding of the same clone in a FAP-targeted monovalent format (filledtriangle) was clearly weaker due to loss of avidity binding. In theabsence of human Ox40 expressing cells no binding can be observed(resting cells, left graphs). Shown is the binding as median offluorescence intensity (MFI) of FITC labeled anti-human IgG Fcγ-specificgoat IgG F(ab′)2 fragment which is used as secondary detection antibody.MFI was measured by flow cytometry and baseline corrected by subtractingthe MFI of the blank control (see Example 4.3.2.1). DP88 huIgG1 P329GLALA is isotype antibody used as control. The x-axis shows theconcentration of antibody constructs. In FIGS. 14A, 14B, 14C and 14D itcan be seen that clone 1G4 binds to activated, OX40 expressing human CD4T cells, and to a lower extent to activated human CD8 T cells. Thebivalent construct binds stronger than the monovalent construct. Theconstructs do not bind to OX40 negative resting T cells. FIGS. 14E, 14F,14G and 14H show that clone 8H9 binds to activated, OX40-expressinghuman CD4 T cells, and to a lower extent to activated human CD8 T cells.The bivalent construct binds stronger than the monovalent construct. Theconstructs do not bind to OX40 negative resting T cells.

FIGS. 14J, 14K, 14L and 14M show that the bivalent FAP-targeted OX40constructs showed stronger binding characteristics to OX40 positivecells as respective clone in a monovalent antibody format.

FIGS. 14N, 14O, 14P and 14Q show that different 2+1 constructs boundwith similar strength to OX40 positive T cells, independently of thesecond binding moiety.

FIGS. 15A, 15B, 15C, 15D, 15E and 15F show the binding of selectedanti-OX40 binders (clone 8H9, 1G4) in a FAP targeted monovalent orbivalent format to FAP positive tumor cells. Transgenic modified mouseembryonic fibroblast NIH/3T3-huFAP clone 39 or WM266-4 cells expresshigh levels of human fibroblast activation protein (huFAP). OnlyFAP-targeted mono- and bivalent anti-Ox40 constructs (filled square andtriangle) but not the same clone in a human IgG1 P329GLALA format (opensquare) binds to NIH/3T3-huFAP clone 39 cells (FIG. 15A) and WM266-4cells (FIG. 15B), respectively. Shown is the binding as median offluorescence intensity (MFI) of Fluorescein isothiocyanate(FITC)-labeled anti-human IgG Fcγ-specific goat IgG F(ab′)2 fragmentwhich is used as secondary detection antibody. MFI was measured by flowcytometry. The x-axis shows the concentration of antibody constructs.The bivalent FAP construct binds stronger than the monovalent construct.Binding to human FAP-expressing tumor cells is also shown in FIGS. 15C,15D, 15E and 15F (see Example 4.5.4.2 for more details).

FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H and 16J, 16K, 16L, 16M and16N show the NFκB activation by selected binders (8H9, 1G4) in amonovalent or bivalent FAP targeted format in the presence or absence ofhyper-crosslinking. Shown is the activation of NF-κB signaling pathwayin the reporter cells by selected binders 1G4 (FIG. 16A-16D) and 8H9(FIG. 16E-16G) in a monovalent (filled triangle) or bivalent (filledsquare) FAP targeted format or as non-targeted hu IgG P329GLALAantibodies (open square). Hyper-crosslinking was provided by eitheranti-hu IgG Fcγ-specific secondary antibodies (ratio 1:2 of primary tosecondary antibodies) or via FAP-expressing NIH/3T3-huFAP clone 39 andWM266-4 tumor cells (ratio 2:1 of FAP⁺ tumor cells to reporter cells).The NF-κB-mediated luciferase activity was characterized by blotting theunits of released light (URL), measured during 0.5 s, versus theconcentration in nM of tested compounds. URLs are emitted due toluciferase-mediated oxidation of luciferin to oxyluciferin. Values arebaseline corrected by subtracting the URLs of the blank control. In FIG.16A it can be seen that all constructs containing clone 1G4 were able toinduce NFkB activation in Ox40⁺ HeLa reporter cells. Crosslinking bysecondary anti IgG Fcγ specific antibody strongly increased NFκBactivation. Addition of FAP positive cells (NIH or WM266-4) howeverincreased only the agonistic potential of FAP targeted molecules, butnot that of the P329GLALA IgG format. Bivalent constructs performedclearly better than monovalent constructs. In FIGS. 16E, 16F and 16G itis shown that all constructs containing clone 8H9 were able to induceNFκB activation in OX40⁺ HeLa reporter cells. Crosslinking by secondaryanti IgG Fcγ specific antibody strongly increased NFκB activation.Addition of FAP positive cells (NIH) however increased only theagonistic potential of FAP targeted molecules, but not that of theP329GLALA IgG format. Bivalent constructs performed slightly better thanmonovalent constructs. In FIGS. 16H, 16J and 16K it can also be seenthat constructs with monovalent binding to OX40 (1+1) are less efficientthan constructs with bivalent binding to OX40 (2+1 and 2+2 constructs.Further data are given in FIGS. 16L, 16M and 16N and in Example 5.1.

FIGS. 17A and 17B show the rescue of suboptimal TCR restimulation ofpreactivated CD4 T cells with plate-immobilized FAP targeted mono andbivalent anti-OX40 (1G4 and 8H9) constructs. Costimulation withplate-immobilized anti-Ox40 binders (huIgG1 P329GLALA format) promotedcell proliferation and maturation of sub-optimally restimulated humanCD4 T cells and induced an enhanced activated phenotype. PHA-Lpre-activated CFSE-labeled human CD4 T cells were cultured for four dayson plates pre-coated with mouse IgG Fcγ spec. antibodies, human IgG Fcγspec. antibodies (both 2 μg/mL), mouse anti-human CD3 antibodies (cloneOKT3, [3 ng/mL]) and titrated anti-Ox40 binders (huIgG1 P329GLALAformat). Shown is the event count, the percentage of proliferating(CFSE-low) cells, the percentage of effector T cells (CD127lowCD45RAlow) and the percentage of CD62L low, OX40 positive or Tim-3positive cells at day 4. Baseline values of samples containing only theplate-immobilized anti-human CD3 were substracted. Therefore, theenhancing effect of OX40 stimulation but not the effect of suboptimalanti-CD3 stimulation per se is visible here. It can be seen that allconstructs containing clone 8H9 were able to rescue suboptimal TCRstimulation of preactivated, Ox40⁺ CD4 T cells when coated to plate.Cells showed a more activated (Tim3⁺ FSC⁺) phenotype. Bivalentconstructs performed slightly better than monovalent constructs.

FIGS. 18A, 18B, 18C and 18D show the rescue of suboptimal TCRrestimulation of preactivated CD4 T cells with plate-immobilized FAPtargeted mono and bivalent anti-OX40 (1G4 and 8H9) constructs.Costimulation with plate-immobilized anti-Ox40 binders (huIgG1 P329GLALAformat) promoted cell proliferation and maturation of sub-optimallyrestimulated human CD4 T cells and induced an enhanced activatedphenotype. PHA-L pre-activated CFSE-labeled human CD4 T cells werecultured for four days on plates pre-coated with mouse IgG Fc□ spec.antibodies, human IgG Fc□ spec. antibodies (both 2 □g/mL), mouseanti-human CD3 antibodies (clone OKT3, [3 ng/mL]) and titrated anti-Ox40binders (huIgG1 P329GLALA format). Shown is the event count, thepercentage of proliferating (CFSE-low) cells, the percentage of effectorT cells (CD127low CD45RAlow) and the percentage of CD62L low, OX40positive or Tim-3 positive cells at day 4. Baseline values of samplescontaining only the plate-immobilized anti-human CD3 were substracted.Therefore, the enhancing effect of OX40 stimulation but not the effectof suboptimal anti-CD3 stimulation per se is visible here. It can beobserved that all constructs containing clone 1G4 were able to rescuesuboptimal TCR stimulation of preactivated, Ox40⁺ CD4 T cells whencoated to plate. Cells proliferate more and present an activated (Tim3⁺Ox40⁺) phenotype. Bivalent constructs perform better than monovalentconstructs. Both bivalent constructs performed comparable when coated toplate.

FIG. 19 the EC₅₀ values as calculated from rescuing suboptimal TCRstimulation with plate-immobilized FAP targeted mono and bivalentanti-OX40 (clone 1G4) constructs are shown. The percentage ofproliferating (CFSE-low) cells and the percentage of CD127L low, Tim-3positive and OX40 positive cells at day 4 were plotted vs the anti-OX40antibody concentration and EC₅₀ values as measure for agonistic strengthwere calculated using the inbuilt sigmoidal dose response quotation inPrism4 (GraphPad Software, USA). All constructs containing clone 1G4were able to rescue suboptimal TCR stimulation of preactivated, Ox40⁺CD4 T cells when coated to plate. However, the bivalent (2+2) constructsperformed better than monovalent (1+1) constructs.

FIGS. 20A, 20B, 20C, 20D, 20E, 20F, 20G and 20H relate to the OX40mediated costimulation of suboptimally TCR triggered resting human PBMCand hypercrosslinking by cell surface FAP. Only constructs containingFAP binding moiety were able to rescue suboptimal TCR stimulation ofpreactivated, Ox40⁺ CD4 T cells when crosslinking was provided by FAPpositive cells (NIH). Shown is either the event count (FIGS. 20B and20D), the percentage of low-proliferating (CFSE-high) cells (FIGS. 20Aand 20C) or the MFI of CD62L (FIGS. 20F and 20H), CD127 (FIG. 20E) orGranzymeB vital CD4⁺ (FIG. 20G) and CD8⁺ T cells. Baseline values ofsamples containing only the anti-human CD3 (clone V9, huIgG1), restinghuman PBMC and NIH/3T3-huFAP clone 39 were substracted. Thus theenhancing effect of OX40 costimulation but not the effect of suboptimalanti-CD3 stimulation per se is shown here. Cells survived better,proliferated more and showed a stronger activated (CD62L and CD127 low)phenotype. Targeted bivalent constructs performed only slightly betterthan monovalent constructs. Clone 8H9 in a non-targeted huIgG1P329GLALAwas not able to rescue suboptimal TCR stimulation in the absence offurther crosslinking. In a FAP positive tumor micro environment thiscould lead to increased anti-tumor activity of T cells whereby systemicOX40 activation is avoided.

FIGS. 21A, 21B and 21C relate to the activation of resting human CD4cell using surface immobilized FAP targeted mono and bivalent anti-OX40(1G4) constructs. Costimulation with non-targeted anti-Ox40 (1G4) huIgG1did not rescue suboptimally TCR stimulated CD4 and CD8 (data not shown)T cells. Hyper-crosslinking of the FAP targeted mono and bivalentanti-Ox40 constructs by the present NIH/3T3-huFAP clone 39 cellsstrongly promoted proliferation, survival (data not shown) and inducedan enhanced activated phenotype in human CD4 cells. Shown is the MFI ofCD25 expression on CD4 T cells and the percentage of CD25⁺ CD4 T cells.Baseline values of samples containing only the anti-human CD3 (clone V9,huIgG1), resting human PBMC and NIH/3T3-huFAP clone 39 were substracted.The agonistic effect of the compounds were quantified as area under thecurve using the inbuilt function in GraphPad Prism and is shown for thethree different anti-Ox40 (1G4) constructs. Targeted bivalent (2+2)constructs performed better than monovalent (1+1) constructs.

FIGS. 21D, 21E, 21F, 21G, 21H and 21J, 21K, 21L, 21M, 21N and 21P showdata as obtained in a second experiment. Monovalent anti-OX40 construct(1+1; filled triangle) was less able to rescue TCR stimulation thanbivalent anti-OX40 targeting constructs (semi-filled circle, filledsquare). The bivalently to FAP binding 2+2 construct was already able atlower concentrations to rescue suboptimal TCR stimulation compared tothe monovalently to FAP binding 2+1 constructs. In the 2+1 format thehigh affinity FAP binding clone 4B9 was clearly superior to the lowaffinity clone 28H1 (FIGS. 21J, 21K, 21L, 21M and 21N). This suggeststhat the EC₅₀ values of the observed bioactivity were driven by thebinding to FAP (2+2>2+1 (4B9)>2+1 (28H1). In FIG. 21P the agonisticcapacity of each construct was quantified for the analyzed markers asarea under the curve and plotted against each other. The observedbioactivity was best for the FAP (28H1) (2+2) construct, followed by theFAP (4B9) (2+1) construct and then the FAP (28H1) (2+1) construct.

FIGS. 22A and 22B shows that anti-Ox40 antibodies containing human IgG1Fc regions can induc lysis of OX40 positive cells. PkH26 labeledHeLa_hOx40_NFkB_Luc1 and freshly isolated NK cells were cocultured at anE to T ratio 3:1 for 24 hours in the presence of OX40 antibodies (humanIgG1 and human IgG1 P329GLALA). The LDH content was analyzed after 4hours using the cytotoxicity detection kit—LDH (Roche, Cat.No.11644793001). After 24 hrs cells were stained with Dapi and wereanalyzed by flow cytometry. The percentage of Dapi positive dead cellswas used to calculate specific lysis. Anti OX40 binders in a human IgGformat bind to Fc receptors on NK cells and induce ADCC of OX40 positivetarget cells. Using the hu IgG1 P329GLALA format instead prevents ADCCof OX40 positive cells (e.g. recently activated T cells).

FIGS. 23A, 23B, 23C and 23D show the binding to resting and activatedhuman T cells of four anti-human 4-1BB-specific clones transferred to ahuIgG1 P329G LALA format (filled diamond: clone 25G7, filled square:clone 12B3, filled star: clone 11D5, pointing-up triangle: clone 9B11)and one anti-mouse 4-1BB specific clone 20G2 transferred to a huIgG1P329G LALA format (pointing down triangle). As negative control anon-4-1BB-specific clone DP47 huIgG1 P329G LALA antibody was used (opengrey circle). The upper panels show binding to resting CD4⁺ T cells(FIG. 23A) and activated CD4⁺ T cells (FIG. 23B), whereas the lowerpanels show binding to resting CD8⁺ T cells (FIG. 23C) and activatedCD8⁺ T cells (FIG. 23D). The binding is characterized by plotting themedian of fluorescence (MFI) of FITC-labeled or PE-labeled anti-humanIgG Fcγ-specific goat IgG F(ab′)₂ fragment that is used as secondarydetection antibody versus the concentration in nM of the tested primaryanti-4-1BB-binding huIgG1 P329G LALA antibodies. MFI was measured byflow cytometry and baseline corrected by subtracting the MFI of theblank control (no primary antibody).

FIGS. 24A, 24B, 24C and 24D show the binding to 4-1BB expressing mouse Tcells. Shown is the binding to resting and activated mouse T cells offour anti-human 4-1BB binding huIgG1 P329G LALA antibody clones (filleddiamond: clone 25G7, filled square: clone 12B3, filled star: clone 11D5,pointing-up triangle: clone 9B11) and one anti-mouse 4-1BB bindinghuIgG1 P329G LALA antibody clone 20G2 (pointing-down triangle). Asnegative control a non-4-1BB binding DP47 huIgG1 P329G LALA antibody wasused (open grey circle). The upper panels show binding to resting mouseCD4⁺ T cells (FIG. 24A) and activated CD4⁺ T cells (FIG. 24B), whereasthe lower panels show binding to resting mouse CD8⁺ T cells (FIG. 24C)and activated CD8⁺ T cells (FIG. 24D). The binding is characterized byplotting the MFI of FITC-labeled anti-human IgG Fcγ-specific goat IgGF(ab′)₂ fragment that is used as secondary detection antibody versus theconcentration in nM of the tested primary anti-4-1BB-binding huIgG1P329G LALA antibodies. MFI was measured by flow cytometry and baselinecorrected by subtracting the MFI of the blank control (no primaryantibody).

FIGS. 25A, 25B, 25C and 25D show the binding of mouse IgGs to 4-1BBexpressing mouse T cells. Shown is the binding to resting and activatedmouse T cells of the anti-mouse 4-1BB binding clone 20G2 transferred tothe formats mouse IgG1 DAPG and mouse IgG1 wildtype (wt). As negativecontrol a commercial non-4-1BB binding mouse IgG1 wt isotype control wasused (open grey circle, BioLegend, Cat.-No. 400153). In the upper panelsbinding to resting CD4⁺ T cells (FIG. 25A) and activated CD4⁺ T cells(FIG. 25B) is shown, whereas in the lower panels binding to resting CD8⁺T cells (FIG. 25C) and activated CD8⁺ T cells (FIG. 25D) is shown. Thebinding is characterized by plotting the median of fluorescence ofintensity (MFI) of FITC-labeled anti-mouse IgG Fcγ-specific goat IgGF(ab′)2 fragment that is used as secondary detection antibody versus theconcentration in nM of the tested primary anti-4-1BB-binding moIgGantibodies. MFI was measured by flow cytometry and baseline corrected bysubtracting the MFI of the blank control (no primary antibody).

FIGS. 26A and 26B show the binding to 4-1BB expressing cynomolgus Tcells. Shown is the binding to activated cynomolgus T cells of fouranti-human 4-1BB binding huIgG1 P329G LALA antibody clones (filleddiamond: clone 25G7, filled square: clone 12B3, filled star: clone 11D5,pointing-up triangle: clone 9B11). As negative control a non-4-1BBbinding DP47 huIgG1 P329G LALA antibody was used (open grey circle).Shown is the binding to activated CD4⁺ T cells (FIG. 26A) and toactivated CD8⁺ T cells (FIG. 26B) respectively. The binding ischaracterized by plotting the median of fluorescence of intensity (MFI)of FITC-labeled anti-human IgG Fcγ-specific goat IgG F(ab′)2 fragmentthat is used as secondary detection antibody versus the concentration innM of the tested primary anti-4-1BB-binding huIgG1 P329G LALAantibodies. MFI was measured by flow cytometry and baseline corrected bysubtracting the MFI of the blank control (no primary antibody).

FIGS. 27A, 27B, 27C, 27D, 27E refer to ligand binding properties of theanti-4-1BB antibodies of the invention as determined by surface plasmonresonance. The interaction between human anti-4-1BB IgG 25G7, 11D5, 9B11and 12B3 and the preformed complex hu4-1BB Ligand/hu4-1BB is shown aswell as the interaction of mouse anti-4-1BB clone 20G2 and the preformedcomplex mu4-1BB Ligand/mu4-1BB.

FIGS. 28A, 28B, 28C, 28D, 28E and 28F relate to competition bindingexperiments. FIGS. 28A, 28B and 28C show the interaction betweenanti-4-1BB IgG clones 12B3, 11D5 and 25G7 and a preformed complex ofclone 9B11 and hu4-1BB. FIGS. 28D, 28E and 28F show the interactionbetween anti-4-1BB IgG clones 12B3, 9B11 and 25G7 and a preformedcomplex of clone 11D5 and hu4-1BB. It can be concluded that anti-4-1BBclones 12B3, 11D5 and 9B11 share a different spatial epitope as 25G7,since the two antibodies can bind simultaneously to human 4-1BB.

FIGS. 29A, 29B, 29C and 29D show the binding of hybrid 4-1BB Fc(kih)variants to anti-4-1BB antibodies, i.e. binding ofhu4-1BBD1/mu4-1BBD2-Fc(kih) and mu4-1BBD1/hu4-1BBD2-Fc(kih) variants toanti-4-1BB antibodies. Underlined is the 4-1BB domain recognized by theantibody.

FIGS. 30A, 30B, 30C and 30D show the binding of anti-human 4-1BBantibodies 11D5, 12B3, 25G7 and 9B11 to human 4-1BB Domain 1. Anti-human4-1BB antibodies 11D5, 12B3 and 9B11 bind human domain 1 containing4-1BB constructs.

FIGS. 31A, 31B, 31C and 31D show functional properties of differentanti-human 4-1BB clones in vitro. Pre-activated human CD8⁺ T cells wereactivated with different concentrations of surface immobilizedanti-human-4-1BB-specific huIgG1 P329G LALA antibodies in the absence ofanti-human CD3 antibody (FIGS. 31A and 31C) or in the presence ofsub-optimal concentration of surface immobilized anti-human CD3 antibody(FIGS. 31B and 31D). Shown is the frequency of IFNγ⁺ (A and B) and TNFα⁺(C and D) CD8⁺ T cells in the total CD8⁺ T cell population versus theconcentration of surface immobilized 4-1BB-binding huIgG1 P329G LALA inpM. In the presence of CD3-stimulation 4-1BB-co-stimulation couldincrease IFNγ (FIG. 31B) and TNFα (FIG. 31D) secretion in aconcentration dependent manner. In the absence of CD3-stimulation,activation of 4-1BB had no effect on IFNγ (FIG. 31A) and TNFα (FIG. 31C)secretion.

FIGS. 32A and 32B show functional properties of anti-mouse 4-1BB clone20G2 in vitro. Mouse splenocytes were incubated in the presence of 0.5ug/mL anti-mouse IgG1 CD3 hamster IgG (clone 145-2C11) and differentconcentration of anti-mouse 4-1BB antibodies (filled black diamond:mouse IgG, open black diamond: mouse IgG DAPG) or fitting isotypecontrols (filled grey circle: mouse IgG1, open grey circle: mouse IgG1DAPG) in solution. The concentration is indicated on the x-axis in nM.Only if the anti-mouse 4-1BB clone 20G2 mouse IgG1 (black diamonds)could be cross-linked via FcR-expressing cells, activation of Granzyme B(FIG. 32A) and Eomesodermin (FIG. 32B) could be increased in aconcentration dependent manner.

FIG. 33 shows functional properties of anti-mouse 4-1BB clone 20G2 invivo. Shown are the results of three mice per group. After treatmentwith anti-mouse 4-1BB clone 20G2 mouse IgG1 (grey bars) CD8⁺ T cells areaccumulating in the liver in total number (a). Further proliferationmarker Ki67 was upregulated in frequency (b) and total number (c) onCD8⁺ T cells. It also induced a positive feedback loop by upregulationof 4-1BB (CD137) in frequency (d) and total number (e) on CD8⁺ T cells.The strongest effect was seen 1 day after third injection. If mice weretreated with anti-mouse 4-1BB clone 20G2 mouse IgG1 DAPG (black bars)crosslinking of antibody was prevented and no 4-1BB activation occurred.Therefore CD8⁺ T cells in the liver were similar in number and phenotypeas in the PBS treated mice (white bars).

FIG. 34A shows a schematic scheme of an exemplary bispecific, bivalentantigen binding molecule of the invention comprising two Fab fragmentsbinding to 4-1BB and two cross-Fab fragments binding to FAP (2+2format).

FIG. 34B shows the setup for the SPR experiments showing simultaneousbinding to immobilized human 4-1BB and human FAP is shown.

FIG. 34C shows a schematic scheme of an exemplary bispecific, monovalentantigen binding molecule of the invention comprising one Fab fragmentbinding to 4-1BB and one cross-Fab fragment binding to FAP (1+1 format).

FIGS. 35A, 35B, 35C and 35D show simultaneous binding of bispecificbivalent anti-4-1BB/anti-FAP constructs is shown in FIGS. 35A-35D. Thebispecific constructs were used as analyte 1 to immobilized human 4-1BBand human FAP was used as analyte 2. All bispecific constructs couldbind simultaneously human 4-1BB and human FAP.

FIGS. 36A and 36B shows exemplary bispecific antigen binding moleculesthat are bivalent anti-4-1BB and monovalent anti-FAP huIgG1 P329GLALA,termed also 2+1 format. The bispecific antigen binding moleculescomprise two Fab fragments binding to 4-1BB and a VH and VL domainbinding to FAP.

FIGS. 37A, 37B and 37C relate to the simultaneous binding of bispecific2+1 anti-4-1BB and anti-FAP constructs. FIG. 37A is a pictogram of theassay setup; FIGS. 37B and 37C show the detected simultaneous binding ofthe bispecific antigen binding molecules in 2+1 format (analyte 1) toimmobilized human 4-1BB and human FAP.

FIGS. 38A, 38B, 38C, 38D, 38E and 38F show the binding to resting CD4⁺(upper panels) and CD8⁺ T cells (lower panels) of thehuman-4-1BB-specific clone 11D5 (FIGS. 38A and 38C), 12B3 (FIGS. 38B and38D) and 25G7 (FIGS. 38E and 38F). Binding is presented as geo mean offluorescence of intensity of secondary detection antibody PE-conjugatedanti-human IgG Fcγ-fragment-specific goat IgG F(ab2′) fragment versusthe concentration of primary 4-1BB-binding antibody. In all blots thenegative control DP47-untargeted huIgG1 P329G LALA was used (open blackcircle, dotted line). None of the constructs showed specific binding toresting human CD4⁺ T cells (FIGS. 38 A, 38B and 38E) or resting CD8⁺ Tcells (FIGS. 38C, 38D and 38F).

FIGS. 39A, 39B, 39C, 39D, 39E and 39F show the binding to activated CD4⁺(upper panels) and CD8⁺ T cells (lower panels) of thehuman-4-1BB-specific clone 11D5 (FIGS. 39A and 39C), 12B3 (FIGS. 39B and39D) and 25G7 (FIGS. 39E and 39F). Binding is shown as geo mean offluorescence of intensity of secondary detection antibody PE-conjugatedanti-human IgG Fcγ-fragment-specific goat IgG F(ab2′) fragment versusthe concentration of primary 4-1BB-binding antibody. In all blots thenegative control DP47-untargeted huIgG1 P329G LALA was used (open blackcircle, dotted line). All constructs bound mainly to activated humanCD8⁺ T cells (FIGS. 39C, 39D and 39F), which display a higher4-1BB-expression than activated human CD4⁺ T cells (FIGS. 39A, 39B and39E).

FIG. 40 summarizes the binding to activated human CD8⁺ T cells ofdifferent clones and formats as area under the curve (AUC) of bindingcurves. The different formats and used anti-FAP binding clones areindicated as pictograms below the graph, the 4-1BB-binding clones areindicated by column pattern: DP47 control molecule in white, 25G7containing molecules in black, if DP47-untargeted in black with whitestripes, clone 11D5 in greyand clone 12B3 in white/black-check.

FIGS. 41A, 41B, 41C, 41D, 41E and 41F show the binding to humanFAP-expressing melanoma cell line WM-266-4 (FIGS. 41A, 41B and 41E) andNIH/3T3-huFAP cone 19 cells (FIGS. 41C, 41D and 41F). Binding is shownas geo mean of fluorescence of intensity of secondary detection antibodyPE-conjugated anti-human IgG Fcγ-fragment-specific goat IgG F(ab2′)fragment versus the concentration of primary 4-1BB-binding antibody.Binding curves using constructs containing 4-1BB-binding clone 11D5 areshown in FIGS. 41A and 41C, with clone 12B3 in FIGS. 41B and 41D andwith clone 25G7 in FIGS. 41E and 41F. In all blots the negative controlDP47-untargeted huIgG1 P329G LALA was used (open black circle, dottedline). Only FAP-targeted formats bind to the FAP-expressing cells andnot their parental anti-4-1BB huIgG1P329G LALA antibodies. Thereforeindependent of the format all shown FAP-targeted molecules feature aFAP-specific targeting property. Depending on the format, FAP-bindingclone and targeting moiety, some molecules possess a betterFAP-targeting property than others.

FIG. 42 summarizes the binding to NIH/3T3-huFAP cells. Shown is the areaunder the curve (AUC) of the binding curves. Used antibody formats areindicated as pictograms under the graph, the 4-1BB-binding clones areindicated by the column color: DP47 control molecule in white, 25G7containing molecules in black, if DP47-untargeted in black with whitestripes, clone 11D5 in greyand clone 12B3 in white/black-check. Thegraph shows that only FAP-targeted molecules but not their 4-1BB-bindingparental huIgG1 P329G LALA nor the DP47-targeted 4-1BB (25G7)-bindingmolecules can bind to FAP-expressing cells.

FIGS. 43A, 43B, 43C, 43D, 43E, 43F, 43G, 43H and 43I show NF-κB-mediatedluciferase activity in the 4-1BB-expressing reporter cell lineHeLa-hu4-1BB-NFkB-luc. Luciferase activity is shown on the y-axis asunits of released light (URLs) versus the added concentration ofagonistic human4-1BB-binding molecules after 6 hours of incubation. InFIGS. 43A, 43D and 43G no FAP-expressing tumor cells were added. InFIGS. 43B, 43E and 43H FAP-expressing human melanoma cell line WM-266-4and in FIGS. 43C, 43F and 43I human FAP-transfected NIH/3T3 cells wereadded in a ratio 5:1 to the reporter cell line and incubated for 6 h.Activation curves using constructs containing 4-1BB-binding clone 11D5are shown in FIGS. 43A, 43B and 43C, with clone 12B3 in FIGS. 43D, 43Eand 43F and with clone 25G7 in FIGS. 43G, 43H and 43I. Only FAP-targetedformats induce a luciferase activity in the presence of FAP-expressingtumor cells. Activation levels depend on the clone, the format and theFAP-expressing tumor cell line.

FIGS. 44A and 44B summarize the NF-κB-mediated luciferase activity inthe 4-1BB-expressing reporter cell line HeLa-hu4-1BB-NFkB-luc in thepresence of NIH/3T3-huFAP cells. Shown is the area under the curve (AUC)of the activation curves in the presence of NIH/3T3-huFAP cells. Usedantibody formats and anti-FAP clones are indicated as pictograms underthe graph, the different agonistic 4-1BB clones are indicated withdifferent column patterns: DP47 control molecule in white, 25G7containing molecules in black, clone 11D5 in grey and clone 12B3 inwhite/black-check. The graph shows that only FAP-targeted molecules caninduce a strong activation above background. Activation levels depend onthe clone, FAP-targeting and the format.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

As used herein, the term “moiety capable of specific binding to a targetcell antigen” refers to a polypeptide molecule that specifically bindsto an antigenic determinant. In one aspect, the antigen binding moietyis able to activate signaling through its target cell antigen. In aparticular aspect, the antigen binding moiety is able to direct theentity to which it is attached (e.g. the TNF family ligand trimer) to atarget site, for example to a specific type of tumor cell or tumorstroma bearing the antigenic determinant. Moieties capable of specificbinding to a target cell antigen include antibodies and fragmentsthereof as further defined herein. In addition, moieties capable ofspecific binding to a target cell antigen include scaffold antigenbinding proteins as further defined herein, e.g. binding domains whichare based on designed repeat proteins or designed repeat domains (seee.g. WO 2002/020565).

In relation to an antibody or fragment thereof, the term “moiety capableof specific binding to a target cell antigen” refers to the part of themolecule that comprises the area which specifically binds to and iscomplementary to part or all of an antigen. A moiety capable of specificantigen binding may be provided, for example, by one or more antibodyvariable domains (also called antibody variable regions). Particularly,a moiety capable of specific antigen binding comprises an antibody lightchain variable region (VL) and an antibody heavy chain variable region(VH). In a particular aspect, the “moiety capable of specific binding toa target cell antigen” is a Fab fragment or a cross-Fab fragment.

The term “moiety capable of specific binding to a costimulatory TNFreceptor family member” refers to a polypeptide molecule thatspecifically binds to a costimulatory TNF receptor family member. In oneaspect, the antigen binding moiety is able to activate signaling througha costimulatory TNF receptor family member. Moieties capable of specificbinding to a target cell antigen include antibodies and fragmentsthereof as further defined herein. In addition, moieties capable ofspecific binding to a costimulatory TNF receptor family member includescaffold antigen binding proteins as further defined herein, e.g.binding domains which are based on designed repeat proteins or designedrepeat domains (see e.g. WO 2002/020565). Particularly, a moiety capableof specific binding to a costimulatory TNF receptor family membercomprises an antibody light chain variable region (VL) and an antibodyheavy chain variable region (VH). In a particular aspect, the “moietycapable of specific binding to a costimulatory TNF receptor familymember” is a Fab fragment or a cross-Fab fragment.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antigen bindingmolecule is able to specifically bind to at least two distinct antigenicdeterminants. Typically, a bispecific antigen binding molecule comprisestwo antigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites specific for onedistinct antigenic determinant in an antigen binding molecule that arespecific for one distinct antigenic determinant. As such, the terms“bivalent”, “tetravalent”, and “hexavalent” denote the presence of twobinding sites, four binding sites, and six binding sites specific for acertain antigenic determinant, respectively, in an antigen bindingmolecule. In particular aspects of the invention, the bispecific antigenbinding molecules according to the invention can be monovalent for acertain antigenic determinant, meaning that they have only one bindingsite for said antigenic determinant or they can be bivalent for acertain antigenic determinant, meaning that they have two binding sitesfor said antigenic determinant.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); and singledomain antibodies. For a review of certain antibody fragments, seeHudson et al., Nat Med 9, 129-134 (2003). For a review of scFvfragments, see e.g. Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region. According to thepresent invention, the term “Fab fragment” also includes “cross-Fabfragments” or “crossover Fab fragments” as defined below.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a cross-Fab molecule are possible andcomprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together anantigen-binding site which binds specifically to an antigen and whereinsaid linker is a polypeptide of at least 30 amino acids. In addition,these x-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin). CTLA-4(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptorexpressed on mainly CD4⁺ T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies (e.g. U.S. Pat. No.7,166,697B1). Evibodies are around the same size as the isolatedvariable region of an antibody (e.g. a domain antibody). For furtherdetails see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633. An affibody is a scaffold derived from Protein A ofStaphylococcus aureus which can be engineered to bind to antigen. Thedomain consists of a three-helical bundle of approximately 58 aminoacids. Libraries have been generated by randomization of surfaceresidues. For further details see Protein Eng. Des. Sel. 2004, 17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived fromthe A-domain scaffold family. The native domains of approximately 35amino acids adopt a defined disulfide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007). A transferrin is a monomeric serum transportglycoprotein. Transferrins can be engineered to bind different targetantigens by insertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include the Trans-body. Forfurther details see J. Biol. Chem 274, 24066-24073 (1999). DesignedAnkyrin Repeat Proteins (DARPins) are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two alpha-helices and a beta-turn. They can be engineeredto bind different target antigens by randomizing residues in the firstalpha-helix and a beta-turn of each repeat. Their binding interface canbe increased by increasing the number of modules (a method of affinitymaturation). For further details see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1. A single-domain antibody is an antibody fragmentconsisting of a single monomeric variable antibody domain. The firstsingle domains were derived from the variable domain of the antibodyheavy chain from camelids (nanobodies or V_(H)H fragments). Furthermore,the term single-domain antibody includes an autonomous human heavy chainvariable domain (aVH) or V_(NAR) fragments derived from sharks.Fibronectin is a scaffold which can be engineered to bind to antigen.Adnectins consists of a backbone of the natural amino acid sequence ofthe 10th domain of the 15 repeating units of human fibronectin type III(FN3). Three loops at one end of the .beta.-sandwich can be engineeredto enable an Adnectin to specifically recognize a therapeutic target ofinterest. For further details see Protein Eng. Des. Sel. 18, 435-444(2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.Peptide aptamers are combinatorial recognition molecules that consist ofa constant scaffold protein, typically thioredoxin (TrxA) which containsa constrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can beengineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

The term “antigen binding domain” or “antigen-binding site” refers tothe part of an antigen binding molecule that comprises the area whichspecifically binds to and is complementary to part or all of an antigen.Where an antigen is large, an antigen binding molecule may only bind toa particular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by, for example, one or morevariable domains (also called variable regions). Preferably, an antigenbinding domain comprises an antibody light chain variable region (VL)and an antibody heavy chain variable region (VH).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸M to 10⁻¹³ M,e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Incertain embodiments, the target cell antigen is an antigen on thesurface of a tumor cell. In one embodiment, target cell antigen isselected from the group consisting of Fibroblast Activation Protein(FAP), Carcinoembryonic Antigen (CEA), Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),CD19, CD20 and CD33. In particular, the target cell antigen isFibroblast Activation Protein (FAP).

The term “Fibroblast activation protein (FAP)”, also known as Prolylendopeptidase FAP or Seprase (EC 3.4.21), refers to any native FAP fromany vertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FAP as well as any form of FAP that results from processingin the cell. The term also encompasses naturally occurring variants ofFAP, e.g., splice variants or allelic variants. In one embodiment, theantigen binding molecule of the invention is capable of specific bindingto human, mouse and/or cynomolgus FAP. The amino acid sequence of humanFAP is shown in UniProt (www.uniprot.org) accession no. Q12884 (version149, SEQ ID NO:84), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2.The extracellular domain (ECD) of human FAP extends from amino acidposition 26 to 760. The amino acid and nucleotide sequences of aHis-tagged human FAP ECD is shown in SEQ ID NOs 85 and 86, respectively.The amino acid sequence of mouse FAP is shown in UniProt accession no.P97321 (version 126, SEQ ID NO:87), or NCBI RefSeq NP_032012.1. Theextracellular domain (ECD) of mouse FAP extends from amino acid position26 to 761. SEQ ID NOs 88 and 89 show the amino acid and nucleotidesequences, respectively, of a His-tagged mouse FAP ECD. SEQ ID NOs 90and 91 show the amino acid and nucleotide sequences, respectively, of aHis-tagged cynomolgus FAP ECD. Preferably, an anti-FAP binding moleculeof the invention binds to the extracellular domain of FAP.

The term “Carcinoembroynic antigen (CEA)”, also known asCarcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5),refers to any native CEA from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human CEA is shown in UniProt accession no.P06731 (version 151, SEQ ID NO:92). The term “Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP)”, also known as ChondroitinSulfate Proteoglycan 4 (CSPG4) refers to any native MCSP from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human MCSPis shown in UniProt accession no. Q6UVK1 (version 103, SEQ ID NO:93).The term “Epidermal Growth Factor Receptor (EGFR)”, also namedProto-oncogene c-ErbB-1 or Receptor tyrosine-protein kinase erbB-1,refers to any native EGFR from any vertebrate source, including mammalssuch as primates (e.g. humans) non-human primates (e.g. cynomolgusmonkeys) and rodents (e.g. mice and rats), unless otherwise indicated.The amino acid sequence of human EGFR is shown in UniProt accession no.P00533 (version 211, SEQ ID NO:94). The term “CD19” refers toB-lymphocyte antigen CD19, also known as B-lymphocyte surface antigen B4or T-cell surface antigen Leu-12 and includes any native CD19 from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human CD19is shown in Uniprot accession no. P15391 (version 160, SEQ ID NO:95).“CD20” refers to B-lymphocyte antigen CD20, also known asmembrane-spanning 4-domains subfamily A member 1 (MS4A1), B-lymphocytesurface antigen B1 or Leukocyte surface antigen Leu-16, and includes anynative CD20 from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human CD20 is shown in Uniprot accession no. P11836 (version149, SEQ ID NO:96). “CD33” refers to Myeloid cell surface antigen CD33,also known as SIGLEC3 or gp67, and includes any native CD33 from anyvertebrate source, including mammals such as primates (e.g. humans)non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The amino acid sequence of human CD33is shown in Uniprot accession no. P20138 (version 157, SEQ ID NO:97).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usuallyextends from an amino acid residue at about position 231 to an aminoacid residue at about position 340. In one embodiment, a carbohydratechain is attached to the CH2 domain. The CH2 domain herein may be anative sequence CH2 domain or variant CH2 domain. The “CH3 domain”comprises the stretch of residues C-terminal to a CH2 domain in an Fcregion (i.e. from an amino acid residue at about position 341 to anamino acid residue at about position 447 of an IgG). The CH3 regionherein may be a native sequence CH3 domain or a variant CH3 domain (e.g.a CH3 domain with an introduced “protuberance” (“knob”) in one chainthereof and a corresponding introduced “cavity” (“hole”) in the otherchain thereof; see U.S. Pat. No. 5,821,333, expressly incorporatedherein by reference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

Fc receptor binding dependent effector functions can be mediated by theinteraction of the Fc-region of an antibody with Fc receptors (FcRs),which are specialized cell surface receptors on hematopoietic cells. Fcreceptors belong to the immunoglobulin superfamily, and have been shownto mediate both the removal of antibody-coated pathogens by phagocytosisof immune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody, via antibody dependent cell mediated cytotoxicity (ADCC) (seee.g. Van de Winkel, J. G. and Anderson, C. L., J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity forimmunoglobulin isotypes: Fc receptors for IgG antibodies are referred toas FcγR. Fc receptor binding is described e.g. in Ravetch, J. V. andKinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., etal., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin.Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76(1998) 231-248.

Cross-linking of receptors for the Fc-region of IgG antibodies (FcγR)triggers a wide variety of effector functions including phagocytosis,antibody-dependent cellular cytotoxicity, and release of inflammatorymediators, as well as immune complex clearance and regulation ofantibody production. In humans, three classes of FcγR have beencharacterized, which are:

-   -   FcγRI (CD64) binds monomeric IgG with high affinity and is        expressed on macrophages, monocytes, neutrophils and        eosinophils. Modification in the Fc-region IgG at least at one        of the amino acid residues E233-G236, P238, D265, N297, A327 and        P329 (numbering according to EU index of Kabat) reduce binding        to FcγRI. IgG2 residues at positions 233-236, substituted into        IgG1 and IgG4, reduced binding to FcγRI by 10³-fold and        eliminated the human monocyte response to antibody-sensitized        red blood cells (Armour, K. L., et al., Eur. J. Immunol.        29 (1999) 2613-2624).    -   FcγRII (CD32) binds complexed IgG with medium to low affinity        and is widely expressed. This receptor can be divided into two        sub-types, FcγRIIA and FcγRIIB FcγRIIA is found on many cells        involved in killing (e.g. macrophages, monocytes, neutrophils)        and seems able to activate the killing process. FcγRIIB seems to        play a role in inhibitory processes and is found on B cells,        macrophages and on mast cells and eosinophils. On B-cells it        seems to function to suppress further immunoglobulin production        and isotype switching to, for example, the IgE class. On        macrophages, FcγRIIB acts to inhibit phagocytosis as mediated        through FcγRIIA. On eosinophils and mast cells the B-form may        help to suppress activation of these cells through IgE binding        to its separate receptor. Reduced binding for FcγRIIA is found        e.g. for antibodies comprising an IgG Fc-region with mutations        at least at one of the amino acid residues E233-G236, P238,        D265, N297, A327, P329, D270, Q295, A327, R292, and K414        (numbering according to EU index of Kabat).    -   FcγRIII (CD16) binds IgG with medium to low affinity and exists        as two types. FcγRIIIA is found on NK cells, macrophages,        eosinophils and some monocytes and T cells and mediates ADCC. Fc        γ RIIIB is highly expressed on neutrophils. Reduced binding to        FcγRIIIA is found e.g. for antibodies comprising an IgG        Fc-region with mutation at least at one of the amino acid        residues E233-G236, P238, D265, N297, A327, P329, D270, Q295,        A327, 5239, E269, E293, Y296, V303, A327, K338 and D376        (numbering according to EU index of Kabat).

Mapping of the binding sites on human IgG1 for Fc receptors, the abovementioned mutation sites and methods for measuring binding to FcγRI andFcγRIIA are described in Shields, R. L., et al. J. Biol. Chem. 276(2001) 6591-6604.

The term “ADCC” or “antibody-dependent cellular cytotoxicity” is afunction mediated by Fc receptor binding and refers to lysis of targetcells by an antibody as reported herein in the presence of effectorcells. The capacity of the antibody to induce the initial stepsmediating ADCC is investigated by measuring their binding to Fcγreceptors expressing cells, such as cells, recombinantly expressingFcγRI and/or FcγRIIA or NK cells (expressing essentially FcγRIIIA) Inparticular, binding to FcγR on NK cells is measured.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The “Tumor Necrosis factor receptor superfamily” or “TNF receptorsuperfamily” currently consists of 27 receptors. It is a group ofcytokine receptors characterized by the ability to bind tumor necrosisfactors (TNFs) via an extracellular cysteine-rich domain (CRD). Thesepseudorepeats are defined by intrachain disulphides generated by highlyconserved cysteine residues within the receptor chains. With theexception of nerve growth factor (NGF), all TNFs are homologous to thearchetypal TNF-alpha. In their active form, the majority of TNFreceptors form trimeric complexes in the plasma membrane. Accordingly,most TNF receptors contain transmembrane domains (TMDs). Several ofthese receptors also contain intracellular death domains (DDs) thatrecruit caspase-interacting proteins following ligand binding toinitiate the extrinsic pathway of caspase activation. Other TNFsuperfamily receptors that lack death domains bind TNFreceptor-associated factors and activate intracellular signalingpathways that can lead to proliferation or differentiation. Thesereceptors can also initiate apoptosis, but they do so via indirectmechanisms. In addition to regulating apoptosis, several TNF superfamilyreceptors are involved in regulating immune cell functions such as Bcell homeostasis and activation, natural killer cell activation, and Tcell co-stimulation. Several others regulate cell type-specificresponses such as hair follicle development and osteoclast development.Members of the TNF receptor superfamily include the following: Tumornecrosis factor receptor 1 (1A) (TNFRSF1A, CD120a), Tumor necrosisfactor receptor 2 (1B) (TNFRSF1B, CD120b), Lymphotoxin beta receptor(LTBR, CD18), OX40 (TNFRSF4, CD134), CD40 (Bp50), Fas receptor (Apo-1,CD95, FAS), Decoy receptor 3 (TR6, M68, TNFRSF6B), CD27 (S152, Tp55),CD30 (Ki-1, TNFRSF8), 4-1BB (CD137, TNFRSF9), DR4 (TRAILR1, Apo-2,CD261, TNFRSF10A), DR5 (TRAILR2, CD262, TNFRSF10B), Decoy Receptor 1(TRAILR3, CD263, TNFRSF10C), Decoy Receptor 2 (TRAILR4, CD264,TNFRSF10D), RANK (CD265, TNFRSF11A), Osteoprotegerin (OCIF, TR1,TNFRSF11B), TWEAK receptor (Fn14, CD266, TNFRSF12A), TACI (CD267,TNFRSF13B), BAFF receptor (CD268, TNFRSF13C), Herpesvirus entry mediator(HVEM, TR2, CD270, TNFRSF14), Nerve growth factor receptor (p75NTR,CD271, NGFR), B-cell maturation antigen (CD269, TNFRSF17),Glucocorticoid-induced TNFR-related (GITR, AITR, CD357, TNFRSF18), TROY(TNFRSF19), DR6 (CD358, TNFRSF21), DR3 (Apo-3, TRAMP, WS-1, TNFRSF25)and Ectodysplasin A2 receptor (XEDAR, EDA2R).

Several members of the tumor necrosis factor receptor (TNFR) familyfunction after initial T cell activation to sustain T cell responses.The term “costimulatory TNF receptor family member” or “costimulatoryTNF family receptor” refers to a subgroup of TNF receptor familymembers, which are able to costimulate proliferation and cytokineproduction of T-cells. The term refers to any native TNF family receptorfrom any vertebrate source, including mammals such as primates (e.g.humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g.mice and rats), unless otherwise indicated. In specific embodiments ofthe invention, costimulatory TNF receptor family members are selectedfrom the group consisting of OX40 (CD134), 4-1BB (CD137), CD27, HVEM(CD270), CD30, and GITR, all of which can have costimulatory effects onT cells. More particularly, the costimulatory TNF receptor family memberis selected from the group consisting of OX40 and 4-1BB.

Further information, in particular sequences, of the TNF receptor familymembers may be obtained from publically accessible databases such asUniprot (www.uniprot.org). For instance, the human costimulatory TNFreceptors have the following amino acid sequences: human OX40 (UniProtaccession no. P43489, SEQ ID NO:98), human 4-1BB (UniProt accession no.Q07011, SEQ ID NO:99), human CD27 (UniProt accession no. P26842, SEQ IDNO:100), human HVEM (UniProt accession no. Q92956, SEQ ID NO:101), humanCD30 (UniProt accession no. P28908, SEQ ID NO:102), and human GITR(UniProt accession no. Q9Y5U5, SEQ ID NO:103).

The term “OX40”, as used herein, refers to any native OX40 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed OX40 as well as any form of OX40that results from processing in the cell. The term also encompassesnaturally occurring variants of OX40, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human OX40 is shown inSEQ ID NO: 98 (Uniprot P43489, version 112) and the amino acid sequenceof an exemplary murine OX40 is shown in SEQ ID NO: 104 (Uniprot P47741,version 101).

The terms “anti-OX40 antibody”, “anti-OX40”, “OX40 antibody and “anantibody that specifically binds to OX40” refer to an antibody that iscapable of binding OX40 with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting OX40. Inone embodiment, the extent of binding of an anti-OX40 antibody to anunrelated, non-OX40 protein is less than about 10% of the binding of theantibody to OX40 as measured, e.g., by a radioimmunoassay (RIA) or flowcytometry (FACS). In certain embodiments, an antibody that binds to OX40has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM,≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁶M or less, e.g. from 10⁻⁶⁸M to10⁻¹³M, e.g., from 10⁻⁸M to 10⁻¹⁰ M).

The term “4-1BB”, as used herein, refers to any native 4-1BB from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed 4-1BB as well as any form of4-1BB that results from processing in the cell. The term alsoencompasses naturally occurring variants of 4-1BB, e.g., splice variantsor allelic variants. The amino acid sequence of an exemplary human 4-1BBis shown in SEQ ID NO: 99 (Uniprot accession no. Q07011), the amino acidsequence of an exemplary murine 4-1BB is shown in SEQ ID NO: 105(Uniprot accession no. P20334) and the amino acid sequence of anexemplary cynomolgous 4-1BB (from Macaca mulatta) is shown in SEQ IDNO:106 (Uniprot accession no. F6W5G6).

The terms “anti-4-1BB antibody”, “anti-4-1BB”, “4-1BB antibody and “anantibody that specifically binds to 4-1BB” refer to an antibody that iscapable of binding 4-1BB with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting 4-1BB.In one embodiment, the extent of binding of an anti-4-1BB antibody to anunrelated, non-4-1BB protein is less than about 10% of the binding ofthe antibody to 4-1BB as measured, e.g., by a radioimmunoassay (RIA) orflow cytometry (FACS). In certain embodiments, an antibody that binds to4-1BB has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁶M or less, e.g. from10⁻⁶⁸M to 10⁻¹³M, e.g., from 10⁻⁸M to 10⁻¹⁰ M).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers, wherein “n” is generally a number between 1 and 10,typically between 2 and 4, in particular 2, i.e. the peptides selectedfrom the group consisting of GGGGS (SEQ ID NO: 107) GGGGSGGGGS (SEQ IDNO:108), SGGGGSGGGG (SEQ ID NO:109) and GGGGSGGGGSGGGG (SEQ ID NO:110),but also include the sequences GSPGSSSSGS (SEQ ID NO:111), (G4S)₃ (SEQID NO:112), (G4S)₄ (SEQ ID NO:113), GSGSGSGS (SEQ ID NO:114), GSGSGNGS(SEQ ID NO:115), GGSGSGSG (SEQ ID NO:116), GGSGSG (SEQ ID NO:117), GGSG(SEQ ID NO:118), GGSGNGSG (SEQ ID NO:119), GGNGSGSG (SEQ ID NO:120) andGGNGSG (SEQ ID NO:121). Peptide linkers of particular interest are (G4S)(SEQ ID NO:107), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:108) and GSPGSSSSGS(SEQ ID NO:111).

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

By “fused” or “connected” is meant that the components (e.g. a heavychain of an antibody and a Fab fragment) are linked by peptide bonds,either directly or via one or more peptide linkers.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain embodiments, amino acid sequence variants of the TNF ligandtrimer-containing antigen binding molecules provided herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the TNF ligandtrimer-containing antigen binding molecules. Amino acid sequencevariants of the TNF ligand trimer-containing antigen binding moleculesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the molecules, or by peptide synthesis.Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of residues within the amino acidsequences of the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics, e.g.,antigen-binding. Sites of interest for substitutional mutagenesisinclude the HVRs and Framework (FRs). Conservative substitutions areprovided in Table B under the heading “Preferred Substitutions” andfurther described below in reference to amino acid side chain classes(1) to (6). Amino acid substitutions may be introduced into the moleculeof interest and the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity, or improvedADCC or CDC.

TABLE B Preferred Original Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includebispecific antigen binding molecules of the invention with an N-terminalmethionyl residue. Other insertional variants of the molecule includethe fusion to the N- or C-terminus to a polypeptide which increases theserum half-life of the bispecific antigen binding molecules.

In certain embodiments, the bispecific antigen binding moleculesprovided herein are altered to increase or decrease the extent to whichthe antibody is glycosylated. Glycosylation variants of the moleculesmay be conveniently obtained by altering the amino acid sequence suchthat one or more glycosylation sites is created or removed. Where theTNF ligand trimer-containing antigen binding molecule comprises an Fcregion, the carbohydrate attached thereto may be altered. Nativeantibodies produced by mammalian cells typically comprise a branched,biantennary oligosaccharide that is generally attached by an N-linkageto Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in TNF family ligandtrimer-containing antigen binding molecule may be made in order tocreate variants with certain improved properties. In one aspect,variants of bispecific antigen binding molecules or antibodies of theinvention are provided having a carbohydrate structure that lacks fucoseattached (directly or indirectly) to an Fc region. Such fucosylationvariants may have improved ADCC function, see e.g. US Patent PublicationNos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko KogyoCo., Ltd). In another aspect, variants of the bispecific antigen bindingmolecules or antibodies of the invention are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region is bisected by GlcNAc. Such variants may have reducedfucosylation and/or improved ADCC function, see for example WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function andare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain aspects, it may be desirable to create cysteine engineeredvariants of the bispecific antigen binding molecules of the invention,e.g., “thioMAbs,” in which one or more residues of the molecule aresubstituted with cysteine residues. In particular aspects, thesubstituted residues occur at accessible sites of the molecule. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certain aspects,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and S400 (EU numbering) of the heavy chain Fcregion. Cysteine engineered antigen binding molecules may be generatedas described, e.g., in U.S. Pat. No. 7,521,541.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Bispecific Antibodies of the Invention

The invention provides novel biospecific antigen binding molecules withparticularly advantageous properties such as producibility, stability,binding affinity, biological activity, targeting efficiency and reducedtoxicity.

Exemplary Bispecific Antigen Binding Molecules

In one aspect, the invention provides bispecific antigen bindingmolecules, comprising

-   -   (a) at least one moiety capable of specific binding to a        costimulatory TNF receptor family member,    -   (b) at least one moiety capable of specific binding to a target        cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In a particular aspect, these bispecific antigen binding molecules arecharacterized by agonistic binding to a costimulatory TNF receptorfamily member. Particularly, the costimulatory TNF receptor familymember is selected from the group consisting of OX40 and 4-1BB.

Bispecific Antigen Binding Molecules Binding to OX40

In one aspect, the costimulatory TNF receptor family member is OX40.Particularly, the invention provides bispecific antigen bindingmolecules, wherein the moiety capable of specific binding to acostimulatory TNF receptor family member binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:1.

In one aspect, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to OX40,wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:2 and SEQ ID NO:3,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:4 and SEQ ID NO:5, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,        SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID        NO:15,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID        NO:18, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

In particular, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to OX40,wherein said moiety comprises

(a) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:6 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:19,

(b) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:20,

(c) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:21,

(d) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:9 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:22,

(e) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:10 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:14, CDR-H2 comprising the amino acid sequence of SEQ ID NO:17 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:23,

(f) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:11 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:14, CDR-H2 comprising the amino acid sequence of SEQ ID NO:17 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:23, or

(g) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:12 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:15, CDR-H2 comprising the amino acid sequence of SEQ ID NO:18 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:24.

In one aspect, the invention provides a bispecific antigen bindingmolecule, comprising at least one moiety capable of specific binding toOX40, wherein said moiety comprises a VH domain comprising CDR-H1comprising the amino acid sequence of SEQ ID NO:2, CDR-H2 comprising theamino acid sequence of SEQ ID NO:4, CDR-H3 comprising the amino acidsequence of SEQ ID NO:7 and a VL domain comprising CDR-L1 comprising theamino acid sequence of SEQ ID NO:13, CDR-H2 comprising the amino acidsequence of SEQ ID NO:16 and CDR-H3 comprising the amino acid sequenceof SEQ ID NO:20.

In another aspect, the invention provides a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to OX40comprises a heavy chain variable region VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35 and SEQ ID NO:37 and a light chain variable regionVL comprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ IDNO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36 and SEQ ID NO:38.

Particularly, provided is a bispecific antigen binding molecule, whereinthe moiety capable of specific binding to OX40 comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and a light chain variable region VL        comprising an amino acid sequence of SEQ ID NO:38.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to OX40comprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:27 and a light chain variable region VL comprisingan amino acid sequence of SEQ ID NO:28.

Bispecific Antigen Binding Molecules Binding to 4-1BB

In another aspect, the costimulatory TNF receptor family member is4-1BB. Particularly, the invention provides bispecific antigen bindingmolecules, wherein the moiety capable of specific binding to acostimulatory TNF receptor family member binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO:39.

In one aspect, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to 4-1BB,wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:40 and SEQ ID NO:41,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:42 and SEQ ID NO:43, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:44, SEQ ID NO:45, SEQ ID        NO:46, SEQ ID NO:47 and SEQ ID NO:48        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:49 and SEQ ID NO:50,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:51 and SEQ ID NO:52, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ ID        NO:55, SEQ ID NO:56 and SEQ ID NO:57.

In particular, provided is a bispecific antigen binding molecule,comprising at least one moiety capable of specific binding to 4-1BB,wherein said moiety comprises

(a) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:44 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:53,

(b) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:41, CDR-H2 comprising the amino acid sequence of SEQ ID NO:43,CDR-H3 comprising the amino acid sequence of SEQ ID NO:45 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:50, CDR-H2 comprising the amino acid sequence of SEQ ID NO:52 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:54,

(c) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:46 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:55,

(d) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:47 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:56, or

(e) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:48 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:57.

In another aspect, the invention provides a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to 4-1BBcomprises a heavy chain variable region VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 and SEQ IDNO:66 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 and SEQ IDNO:67.

Particularly, provided is a bispecific antigen binding molecule, whereinthe moiety capable of specific binding to 4-1BB comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

The bispecific antigen binding molecules of the invention are furthercharacterized by comprising at least one moiety capable of specificbinding to a target cell antigen. The bispecific antigen bindingmolecules thus possess the advantage over conventional antibodiescapable of specific binding to a costimulatory TNF receptor familymember, that they selectively induce a costimulatory T cell response atthe target cells, which are typically cancer cells. In one aspect, thetarget cell antigen is selected from the group consisting of FibroblastActivation Protein (FAP), Melanoma-associated Chondroitin SulfateProteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.

Bispecific Antigen Binding Molecules Wherein the Target Cell Antigen isFAP

In a particular aspect, the target cell antigen is Fibroblast ActivationProtein (FAP). FAP binding moieties have been described in WO 2012/02006which is included by reference in its entirety. FAP binding moieties ofparticular interest are described below.

In one aspect, the invention provides a bispecific antigen bindingmolecule, wherein the moiety capable of specific binding to FAPcomprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:68 and SEQ ID NO:69,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:70 and SEQ ID NO:71, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:72 and SEQ ID NO:73,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:74 and SEQ ID NO:75,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:76 and SEQ ID NO:77, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:78 and SEQ ID NO:79.

In particular, provided is a bispecific antigen binding molecule,wherein the moiety capable of specific binding to FAP comprises

(a) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:68, CDR-H2 comprising the amino acid sequence of SEQ ID NO:70,CDR-H3 comprising the amino acid sequence of SEQ ID NO:72 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:74, CDR-H2 comprising the amino acid sequence of SEQ ID NO:76 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:78, or

(b) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:69, CDR-H2 comprising the amino acid sequence of SEQ ID NO:71,CDR-H3 comprising the amino acid sequence of SEQ ID NO:73 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:75, CDR-H2 comprising the amino acid sequence of SEQ ID NO:77 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:79.

In a particular aspect, the moiety capable of specific binding to FAPcomprises a VH domain comprising CDR-H1 comprising the amino acidsequence of SEQ ID NO:69, CDR-H2 comprising the amino acid sequence ofSEQ ID NO:71, CDR-H3 comprising the amino acid sequence of SEQ ID NO:73and a VL domain comprising CDR-L1 comprising the amino acid sequence ofSEQ ID NO:75, CDR-H2 comprising the amino acid sequence of SEQ ID NO:77and CDR-H3 comprising the amino acid sequence of SEQ ID NO:79.

Particularly, provided is a bispecific antigen binding molecule, whereinthe moiety capable of specific binding to FAP comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:80 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:81, or    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:82 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:83.

Bispecific Antigen Binding Molecules Binding to OX40 and FAP

In a further aspect, provided is a bispecific antigen binding molecule,wherein

-   (i) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29,    SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID    NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein

-   (a) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:25 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:26 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (b) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:25 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:26 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (c) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:27 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:28 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (d) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:27 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:28 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (e) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:29 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:30 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (f) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:29 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:30 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (g) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:31 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:32 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (h) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:31 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:32 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (i) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:33 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:34 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (j) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:33 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:34 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (k) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:35 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:36 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (l) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:35 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:36 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (m) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:37 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:38 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81, or-   (n) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence of SEQ ID    NO:37 and a light chain variable region comprising an amino acid    sequence of SEQ ID NO:38 and the moiety capable of specific binding    to FAP comprises a heavy chain variable region VH comprising an    amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83.

In a particular aspect, the invention provides a bispecific antigenbinding molecule, wherein the moiety capable of specific binding to OX40comprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:27 and a light chain variable region comprising anamino acid sequence of SEQ ID NO:28 and the moiety capable of specificbinding to FAP comprises a heavy chain variable region VH comprising anamino acid sequence of SEQ ID NO:80 and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:81, or wherein the moietycapable of specific binding to OX40 comprises a heavy chain variableregion VH comprising an amino acid sequence of SEQ ID NO:27 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:28and the moiety capable of specific binding to FAP comprises a heavychain variable region VH comprising an amino acid sequence of SEQ IDNO:82 and a light chain variable region comprising an amino acidsequence of SEQ ID NO:83.

Bispecific Antigen Binding Molecules Binding to 4-1BB and FAP

In another aspect, provided is a bispecific antigen binding molecule,wherein

-   (i) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,    SEQ ID NO:64 or SEQ ID NO:66 and a light chain variable region    comprising an amino acid sequence that is at least about 95%, 96%,    97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID    NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein

-   (a) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:58 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:59 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (b) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:58 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:95 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (c) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:60 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:61 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (d) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:60 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:61 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (e) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:62 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:63 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (f) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:62 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:63 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (g) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:64 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:65 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81,-   (h) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:64 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:65 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83,-   (i) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:66 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:67 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:80 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:81, or-   (j) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence of    SEQ ID NO:66 and a light chain variable region comprising an amino    acid sequence of SEQ ID NO:67 and the moiety capable of specific    binding to FAP comprises a heavy chain variable region VH comprising    an amino acid sequence of SEQ ID NO:82 and a light chain variable    region comprising an amino acid sequence of SEQ ID NO:83.

Bispecific, Monovalent Antigen Binding Molecules (1+1 Format)

In one aspect, the invention relates to bispecific antigen bindingmolecules comprising (a) one moiety capable of specific binding to acostimulatory TNF receptor family member, (b) one moiety capable ofspecific binding to a target cell antigen, and (c) a Fc domain composedof a first and a second subunit capable of stable association.

In a particular aspect, provided is a bispecific antigen bindingmolecule, wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to a        costimulatory TNF receptor family member,    -   (b) a second Fab fragment capable of specific binding to a        target cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In one aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to OX40,    -   (b) a second Fab fragment capable of specific binding to a        target cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In a further aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to OX40,    -   (b) a second Fab fragment capable of specific binding to FAP,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:303, a first light chain comprising the amino acid sequence of    SEQ ID NO:182, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (b) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:231, a first light chain comprising the amino acid sequence of    SEQ ID NO: 186, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (c) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:233, a first light chain comprising the amino acid sequence of    SEQ ID NO:190, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (d) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:235, a first light chain comprising the amino acid sequence of    SEQ ID NO:194, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (e) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 237, a first light chain comprising the amino acid sequence of    SEQ ID NO:198, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (f) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:239, a first light chain comprising the amino acid sequence of    SEQ ID NO:202, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217.

In one aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to 4-1BB,    -   (b) a second Fab fragment capable of specific binding to a        target cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In a further aspect, provided is a bispecific antigen binding molecule,wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to 4-1BB,    -   (b) a second Fab fragment capable of specific binding to FAP,        and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:295, a first light chain comprising the amino acid sequence of    SEQ ID NO:261, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (b) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:297, a first light chain comprising the amino acid sequence of    SEQ ID NO:265, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (c) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:299, a first light chain comprising the amino acid sequence of    SEQ ID NO:269, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217, or-   (d) a first heavy chain comprising the amino acid sequence of SEQ ID    NO:301, a first light chain comprising the amino acid sequence of    SEQ ID NO:273, a second heavy chain comprising the amino acid    sequence of SEQ ID NO:229, and a second light chain comprising the    amino acid sequence of SEQ ID NO:217.

Bispecific, Bivalent Antigen Binding Molecules (2+2 Format)

In another aspect, the invention relates to a bispecific antigen bindingmolecule, comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) two moieties capable of specific binding to a target cell    antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

In one aspect, the bispecific antigen binding molecule is bivalent bothfor the costimulatory TNF receptor family member and for the target cellantigen.

In one aspect, the bispecific antigen binding molecule of the inventioncomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) two additional Fab fragments capable of specific binding to a    target cell antigen, wherein said additional Fab fragments are each    connected via a peptide linker to the C-terminus of the heavy chains    of (a).

In a particular aspect, the peptide linker is (G4S)₄.

In another aspect, the two additional Fab fragments capable of specificbinding to a target cell antigen are crossover Fab fragments wherein thevariable domains VL and VH are replaced by each other and the VL-CHchains are each connected via a peptide linker to the C-terminus of theheavy chains of (a).

In particular, the invention relates to bispecific antigen bindingmolecules, wherein the two Fab fragments capable of specific binding toa costimulatory TNF receptor family member are two Fab fragments capableof specific binding to OX40 or 4-1BB and the two additional Fabfragments capable of specific binding to a target cell antigen arecrossover Fab fragments capable of specific binding to FAP.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two moieties capable of specific binding toOX40, (b) two moieties capable of specific binding to FAP, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:216, a first light chain comprising the amino acid sequence of    SEQ ID NO:182, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (b) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:219, a first light chain comprising the amino acid sequence of    SEQ ID NO:186, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (c) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:221, a first light chain comprising the amino acid sequence of    SEQ ID NO:190, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (d) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:223, a first light chain comprising the amino acid sequence of    SEQ ID NO:194, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (e) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:225, a first light chain comprising the amino acid sequence of    SEQ ID NO:198, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (f) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:227, a first light chain comprising the amino acid sequence of    SEQ ID NO:202, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two moieties capable of specific binding to4-1BB, (b) two moieties capable of specific binding to FAP, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:287, a first light chain comprising the amino acid sequence of    SEQ ID NO:261, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (b) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:289, a first light chain comprising the amino acid sequence of    SEQ ID NO:265, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (c) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:291, a first light chain comprising the amino acid sequence of    SEQ ID NO:269, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217, or-   (d) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO:293, a first light chain comprising the amino acid sequence of    SEQ ID NO:273, and a second light chain comprising the amino acid    sequence of SEQ ID NO:217.

Bispecific Antigen Binding Molecules Bivalent for Binding to aCostimulatory TNF Receptor Family Member and Monovalent for Binding to aTarget Cell Antigen (2+1 Format)

In another aspect, the invention provides a bispecific antigen bindingmolecule comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) one moiety capable of specific binding to a target cell antigen,    and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

Thus, provided is a bispecific antigen binding molecule, wherein thebispecific antigen binding molecule is bivalent for the costimulatoryTNF receptor family member and monovalent for the target cell antigen.

In a particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of one of the heavy chains and wherein the VL domain    is connected via a peptide linker to the C-terminus of the second    heavy chain.

In another particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of the Fc knob heavy chain and wherein the VL domain    is connected via a peptide linker to the C-terminus of the Fc hole    heavy chain.

In another particular aspect, the bispecific antigen binding moleculecomprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of the Fc hole heavy chain and wherein the VL domain    is connected via a peptide linker to the C-terminus of the Fc knob    heavy chain. In particular, the invention relates to bispecific    antigen binding molecules, wherein the two Fab fragments capable of    specific binding to a costimulatory TNF receptor family member are    two Fab fragments capable of specific binding to OX40 or 4-1BB and    the VH and VL domain capable of specific binding to a target cell    antigen are capable of specific binding to FAP.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two Fab fragments capable of specific bindingto OX40, (b) a VH and a VL domain capable of specific binding to FAP,and (c) a Fc domain composed of a first and a second subunit capable ofstable association.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains, each comprising the amino acid sequence of SEQ    ID NO:186, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:306, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:307, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:186, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:310, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:311.

In one aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two Fab fragments capable of specific bindingto 4-1BB, (b) a VH and a VL domain capable of specific binding to FAP,and (c) a Fc domain composed of a first and a second subunit capable ofstable association.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

-   (a) two light chains, each comprising the amino acid sequence of SEQ    ID NO:261, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:318, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:319, or-   (b) two light chains, each comprising the amino acid sequence of SEQ    ID NO:265, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:322, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:323, or-   (c) two light chains, each comprising the amino acid sequence of SEQ    ID NO:269, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:326, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:327, or-   (d) two light chains, each comprising the amino acid sequence of SEQ    ID NO:273, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:330, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:331, or-   (e) two light chains, each comprising the amino acid sequence of SEQ    ID NO:261, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:334, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:335, or-   (f) two light chains, each comprising the amino acid sequence of SEQ    ID NO:265, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:338, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:339, or-   (g) two light chains, each comprising the amino acid sequence of SEQ    ID NO:269, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:342, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:343, or-   (h) two light chains, each comprising the amino acid sequence of SEQ    ID NO:273, a first heavy chain comprising the amino acid sequence of    SEQ ID NO:346, and a second heavy chain comprising the amino acid    sequence of SEQ ID NO:347.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The bispecific antigen binding molecules of the invention furthercomprise a Fc domain composed of a first and a second subunit capable ofstable association.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

The Fc domain confers favorable pharmacokinetic properties to thebispecific antibodies of the invention, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibodies ofthe invention to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Accordingly, in particular embodimentsthe Fc domain of the bispecific antibodies of the invention exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG Fc domain, in particular an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is anIgG1 Fc domain.

In one such aspect the Fc domain (or the bispecific antigen bindingmolecule of the invention comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG1 Fc domain (or the bispecific antigen bindingmolecule of the invention comprising a native IgG1 Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG1 Fc domain (or the bispecific antigen binding moleculeof the invention comprising a native IgG1 Fc domain). In one aspect, theFc domain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular aspect the Fc receptoris an Fcγ receptor. In one aspect, the Fc receptor is a human Fcreceptor. In one aspect, the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. In one aspect, the Fc receptor is aninhibitory Fc receptor. In a specific aspect, the Fc receptor is aninhibitory human Fcγ receptor, more specifically human FcγRIIB. In oneaspect the effector function is one or more of CDC, ADCC, ADCP, andcytokine secretion. In a particular aspect, the effector function isADCC. In one aspect, the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG1 Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the bispecific antigen binding molecule of theinvention comprising said Fc domain) exhibits greater than about 70%,particularly greater than about 80%, more particularly greater thanabout 90% of the binding affinity of a native IgG1 Fc domain (or thebispecific antigen binding molecule of the invention comprising a nativeIgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In a particular aspect, the Fcdomain of the bispecific antigen binding molecule of the inventioncomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one aspect, the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In another aspect, the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In one aspect, the bispecific antigenbinding molecule of the invention comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared tobispecific antibodies of the invention comprising a non-engineered Fcdomain. In a particular aspect, the Fc receptor is an Fcγ receptor. Inother aspects, the Fc receptor is a human Fc receptor. In one aspect,the Fc receptor is an inhibitory Fc receptor. In a specific aspect, theFc receptor is an inhibitory human Fcγ receptor, more specifically humanFcγRIIB. In some aspects the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. Preferably, binding to each of thesereceptors is reduced. In some aspects, binding affinity to a complementcomponent, specifically binding affinity to C1q, is also reduced. In oneaspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) exhibits greater than about 70% of thebinding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule of the invention comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or thebispecific antigen binding molecule of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments the Fc domain of thebispecific antigen binding molecule of the invention is engineered tohave reduced effector function, as compared to a non-engineered Fcdomain. The reduced effector function can include, but is not limitedto, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). Certain antibody variants with improved or diminishedbinding to FcRs are described. (e.g. U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In one aspect of the invention, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329. Insome aspects, the Fc domain comprises the amino acid substitutions L234Aand L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fcdomain, particularly a human IgG1 Fc domain. In one aspect, the Fcdomain comprises an amino acid substitution at position P329. In a morespecific aspect, the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionselected from the group consisting of E233P, L234A, L235A, L235E, N297A,N297D or P331S. In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). The“P329G LALA” combination of amino acid substitutions almost completelyabolishes Fcγ receptor binding of a human IgG1 Fc domain, as describedin PCT Patent Application No. WO 2012/130831 A1. Said document alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions. such antibody is an IgG1 with mutations L234A and L235A orwith mutations L234A, L235A and P329G (numbering according to EU indexof Kabat et al, Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991).

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specificembodiment, the Fc domain is an IgG4 Fc domain comprising an amino acidsubstitution at position 5228 (Kabat numbering), particularly the aminoacid substitution S228P. In a more specific embodiment, the Fc domain isan IgG4 Fc domain comprising amino acid substitutions L235E and S228Pand P329G. This amino acid substitution reduces in vivo Fab arm exchangeof IgG4 antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor. Effector function of an Fc domain, or bispecific antibodies ofthe invention comprising an Fc domain, can be measured by methods knownin the art. A suitable assay for measuring ADCC is described herein.Other examples of in vitro assays to assess ADCC activity of a moleculeof interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al.Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96® non-radioactivecytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

The following section describes preferred aspects of the bispecificantigen binding molecules of the invention comprising Fc domainmodifications reducing Fc receptor binding and/or effector function. Inone aspect, the invention relates to the bispecific antigen bindingmolecule (a) at least one moiety capable of specific binding to acostimulatory TNF receptor family member, (b) at least one moietycapable of specific binding to a target cell antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the Fc domain comprises one or more amino acidsubstitution that reduces the binding affinity of the antibody to an Fcreceptor, in particular towards Fcγ receptor. In another aspect, theinvention relates to the bispecific antigen binding molecule comprising(a) at least one moiety capable of specific binding to a costimulatoryTNF receptor family member, (b) at least one moiety capable of specificbinding to a target cell antigen, and (c) a Fc domain composed of afirst and a second subunit capable of stable association, wherein the Fcdomain comprises one or more amino acid substitution that reduceseffector function. In particular aspect, the Fc domain is of human IgG1subclass with the amino acid mutations L234A, L235A and P329G (numberingaccording to Kabat EU index).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules of the invention comprisedifferent antigen-binding sites, fused to one or the other of the twosubunits of the Fc domain, thus the two subunits of the Fc domain may becomprised in two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the bispecific antibodies of the invention inrecombinant production, it will thus be advantageous to introduce in theFc domain of the bispecific antigen binding molecules of the invention amodification promoting the association of the desired polypeptides.

Accordingly, in particular aspects the invention relates to thebispecific antigen binding molecule comprising (a) at least one moietycapable of specific binding to a costimulatory TNF receptor familymember, (b) at least one moiety capable of specific binding to a targetcell antigen, and (c) a Fc domain composed of a first and a secondsubunit capable of stable association, wherein the Fc domain comprises amodification promoting the association of the first and second subunitof the Fc domain. The site of most extensive protein-protein interactionbetween the two subunits of a human IgG Fc domain is in the CH3 domainof the Fc domain. Thus, in one aspect said modification is in the CH3domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to thebispecific antigen binding molecule comprising (a) at least one moietycapable of specific binding to a costimulatory TNF receptor familymember, (b) at least one moiety capable of specific binding to a targetcell antigen, and (c) a Fc domain composed of a first and a secondsubunit capable of stable association, wherein the first subunit of theFc domain comprises knobs and the second subunit of the Fc domaincomprises holes according to the knobs into holes method. In aparticular aspect, the first subunit of the Fc domain comprises theamino acid substitutions S354C and T366W (EU numbering) and the secondsubunit of the Fc domain comprises the amino acid substitutions Y349C,T366S and Y407V (numbering according to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

The C-terminus of the heavy chain of the bispecific antibody as reportedherein can be a complete C-terminus ending with the amino acid residuesPGK. The C-terminus of the heavy chain can be a shortened C-terminus inwhich one or two of the C terminal amino acid residues have beenremoved. In one preferred aspect, the C-terminus of the heavy chain is ashortened C-terminus ending PG. In one aspect of all aspects as reportedherein, a bispecific antibody comprising a heavy chain including aC-terminal CH3 domain as specified herein, comprises the C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to Kabat EUindex). In one embodiment of all aspects as reported herein, abispecific antibody comprising a heavy chain including a C-terminal CH3domain, as specified herein, comprises a C-terminal glycine residue(G446, numbering according to Kabat EU index).

Modifications in the Fab Domains

In one aspect, the invention relates to a bispecific antigen bindingmolecule comprising (a) a first Fab fragment capable of specific bindingto a costimulatory TNF receptor family member, (b) a second Fab fragmentcapable of specific binding to a target cell antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein in one of the Fab fragments either the variabledomains VH and VL or the constant domains CH1 and CL are exchanged. Thebispecific antibodies are prepared according to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. Theyclearly reduce the byproducts caused by the mismatch of a light chainagainst a first antigen with the wrong heavy chain against the secondantigen (compared to approaches without such domain exchange).

In one aspect, the invention relates to a bispecific antigen bindingmolecule comprising (a) a first Fab fragment capable of specific bindingto a costimulatory TNF receptor family member, (b) a second Fab fragmentcapable of specific binding to a target cell antigen, and (c) a Fcdomain composed of a first and a second subunit capable of stableassociation, wherein in one of the Fab fragments the constant domains CLand CH1 are replaced by each other so that the CH1 domain is part of thelight chain and the CL domain is part of the heavy chain. Moreparticularly, in the second Fab fragment capable of specific binding toa target cell antigen the constant domains CL and CH1 are replaced byeach other so that the CH1 domain is part of the light chain and the CLdomain is part of the heavy chain.

In a particular aspect, the invention relates a bispecific antigenbinding molecule comprising (a) a first Fab fragment capable of specificbinding to a costimulatory TNF receptor family member, (b) a second Fabfragment capable of specific binding to a target cell antigen, whereinthe constant domains CL and CH1 are replaced by each other so that theCH1 domain is part of the light chain and the CL domain is part of theheavy chain. Such a molecule is called a monvalent bispecific antigenbinding molecule.

In another aspect, the invention relates to a bispecific antigen bindingmolecule, comprising (a) two light chains and two heavy chains of anantibody comprising two Fab fragments capable of specific binding to acostimulatory TNF receptor family member and the Fc domain, and (b) twoadditional Fab fragments capable of specific binding to a target cellantigen, wherein said additional Fab fragments are each connected via apeptide linker to the C-terminus of the heavy chains of (a). In aparticular aspect, the additional Fab fragments are Fab fragments,wherein the variable domains VL and VH are replaced by each other sothat the VH domain is part of the light chain and the VL domain is partof the heavy chain.

Thus, in a particular aspect, the invention comprises a bispecific,antigen binding molecule, comprising (a) two light chains and two heavychains of an antibody comprising two Fab fragments capable of specificbinding to a costimulatory TNF receptor family member and the Fc domain,and (b) two additional Fab fragments capable of specific binding to atarget cell antigen, wherein said two additional Fab fragments capableof specific binding to a target cell antigen are crossover Fab fragmentswherein the variable domains VL and VH are replaced by each other andthe VL-CH chains are each connected via a peptide linker to theC-terminus of the heavy chains of (a).

In another aspect, and to further improve correct pairing, thebispecific antigen binding molecule comprising (a) a first Fab fragmentcapable of specific binding to a costimulatory TNF receptor familymember, (b) a second Fab fragment capable of specific binding to atarget cell antigen, and (c) a Fc domain composed of a first and asecond subunit capable of stable association, can contain differentcharged amino acid substitutions (so-called “charged residues”). Thesemodifications are introduced in the crossed or non-crossed CH1 and CLdomains. In a particular aspect, the invention relates to a bispecificantigen binding molecule, wherein in one of CL domains the amino acid atposition 123 (EU numbering) has been replaced by arginine (R) and theamino acid at position 124 (EU numbering) has been substituted by lysine(K) and wherein in one of the CH1 domains the amino acids at position147 (EU numbering) and at position 213 (EU numbering) have beensubstituted by glutamic acid (E).

More particularly, the invention relates to a bispecific bindingmolecule comprising a Fab, wherein in the CL domain adjacent to the TNFligand family member the amino acid at position 123 (EU numbering) hasbeen replaced by arginine (R) and the amino acid at position 124 (EUnumbering) has been substituted by lysine (K), and wherein in the CH1domain adjacent to the TNF ligand family member the amino acids atposition 147 (EU numbering) and at position 213 (EU numbering) have beensubstituted by glutamic acid (E).

Exemplary Antibodies of the Invention

In one aspect, the invention provides new antibodies and antibodyfragments that specifically bind to OX40. These antibodies have superiorproperties compared to known OX40 antibodies that make them especiallysuitable for the incorporation into bispecific antigen binding moleculescomprising another antigen binding moiety capable of specific binding toa target cell antigen.

In particular, provided is an antibody that specifically binds to OX40,wherein said antibody comprises

(a) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:6 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:19,

(b) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:7 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:20,

(c) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:8 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:21,

(d) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:2, CDR-H2 comprising the amino acid sequence of SEQ ID NO:4,CDR-H3 comprising the amino acid sequence of SEQ ID NO:9 and a VL domaincomprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:13,CDR-H2 comprising the amino acid sequence of SEQ ID NO:16 and CDR-H3comprising the amino acid sequence of SEQ ID NO:22,

(e) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:10 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:14, CDR-H2 comprising the amino acid sequence of SEQ ID NO:17 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:23,

(f) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:11 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:14, CDR-H2 comprising the amino acid sequence of SEQ ID NO:17 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:23, or

(g) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:3, CDR-H2 comprising the amino acid sequence of SEQ ID NO:5,CDR-H3 comprising the amino acid sequence of SEQ ID NO:12 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:15, CDR-H2 comprising the amino acid sequence of SEQ ID NO:18 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:24.

In one aspect, provided is an antibody that specifically binds to OX40,wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

In a further aspect, provided is an antibody that competes for bindingwith an antibody that specifically binds to OX40, wherein said antibodycomprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

In one aspect, provided is an antibody that competes for binding with anantibody that specifically binds to OX40, wherein said antibodycomprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:27 and a light chain variable region VL comprisingan amino acid sequence of SEQ ID NO:28. In particular, provided is anantibody that specifically binds to OX40, wherein said antibodycomprises a heavy chain variable region VH comprising an amino acidsequence of SEQ ID NO:27 and a light chain variable region VL comprisingan amino acid sequence of SEQ ID NO:28.

In a further aspect, provided is an antibody that specifically binds toOX40 and is cross-reactive for human and murine OX40, wherein saidantibody comprises a heavy chain variable region VH comprising an aminoacid sequence of SEQ ID NO:31 and a light chain variable region VLcomprising an amino acid sequence of SEQ ID NO:32.

In another aspect, the invention provides new antibodies and antibodyfragments that specifically bind to 4-1BB. These antibodies havesuperior properties compared to known 4-1BB antibodies so that they areespecially suitable for the incorporation into bispecific antigenbinding molecules comprising another antigen binding moiety capable ofspecific binding to a target cell antigen.

In particular, provided is an antibody that specifically binds to 4-1BB,wherein said antibody comprises

(a) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:44 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:53,

(b) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:41, CDR-H2 comprising the amino acid sequence of SEQ ID NO:43,CDR-H3 comprising the amino acid sequence of SEQ ID NO:45 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:50, CDR-H2 comprising the amino acid sequence of SEQ ID NO:52 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:54,

(c) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:46 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:55,

(d) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:47 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:56, or

(e) a VH domain comprising CDR-H1 comprising the amino acid sequence ofSEQ ID NO:40, CDR-H2 comprising the amino acid sequence of SEQ ID NO:42,CDR-H3 comprising the amino acid sequence of SEQ ID NO:48 and a VLdomain comprising CDR-L1 comprising the amino acid sequence of SEQ IDNO:49, CDR-H2 comprising the amino acid sequence of SEQ ID NO:51 andCDR-H3 comprising the amino acid sequence of SEQ ID NO:57.

In one aspect, the invention provides an antibody that specificallybinds to 4-1BB, wherein said antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

In a further aspect, the invention provides an antibody that competesfor binding with an antibody that specifically binds to 4-1BB, whereinsaid antibody comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

Polynucleotides

The invention further provides isolated polynucleotides encoding abispecific antigen binding molecule as described herein or a fragmentthereof.

The isolated polynucleotides encoding bispecific antibodies of theinvention may be expressed as a single polynucleotide that encodes theentire antigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional antigen bindingmolecule. For example, the light chain portion of an immunoglobulin maybe encoded by a separate polynucleotide from the heavy chain portion ofthe immunoglobulin. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes a polypeptidecomprised in the bispecific molecule according to the invention asdescribed herein.

In one aspect, the present invention is directed to an isolatedpolynucleotide encoding a bispecific antigen binding molecule,comprising (a) at least one moiety capable of specific binding to OX40,(b) at least one moiety capable of specific binding to a target cellantigen, and (c) a Fc domain composed of a first and a second subunitcapable of stable association.

In another aspect, provided is an isolated polynucleotide encoding abispecific antigen binding molecule, comprising (a) at least one moietycapable of specific binding to 4-1BB, (b) at least one moiety capable ofspecific binding to a target cell antigen, and (c) a Fc domain composedof a first and a second subunit capable of stable association.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence that encodes an antibody orantibody fragment that specifically binds at OX40.

In another aspect, provided is an isolated polynucleotide encoding anisolated polynucleotide comprising a sequence that encodes an antibodyor antibody fragment that specifically binds at 4-1BB.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Bispecific antibodies of the invention may be obtained, for example, bysolid-state peptide synthesis (e.g. Merrifield solid phase synthesis) orrecombinant production. For recombinant production one or morepolynucleotide encoding the bispecific antigen binding molecule orpolypeptide fragments thereof, e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such polynucleotide may be readily isolated andsequenced using conventional procedures. In one aspect of the invention,a vector, preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing the coding sequence of the bispecific antigen bindingmolecule (fragment) along with appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See, for example, the techniques described in Maniatis etal., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience,N.Y. (1989). The expression vector can be part of a plasmid, virus, ormay be a nucleic acid fragment. The expression vector includes anexpression cassette into which the polynucleotide encoding thebispecific antigen binding molecule or polypeptide fragments thereof(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the bispecificantigen binding molecule of the invention or polypeptide fragmentsthereof, or variants or derivatives thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription.

Suitable promoters and other transcription control regions are disclosedherein. A variety of transcription control regions are known to thoseskilled in the art. These include, without limitation, transcriptioncontrol regions, which function in vertebrate cells, such as, but notlimited to, promoter and enhancer segments from cytomegaloviruses (e.g.the immediate early promoter, in conjunction with intron-A), simianvirus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Roussarcoma virus). Other transcription control regions include thosederived from vertebrate genes such as actin, heat shock protein, bovinegrowth hormone and rabbit â-globin, as well as other sequences capableof controlling gene expression in eukaryotic cells. Additional suitabletranscription control regions include tissue-specific promoters andenhancers as well as inducible promoters (e.g. promoters inducibletetracyclins). Similarly, a variety of translation control elements areknown to those of ordinary skill in the art. These include, but are notlimited to ribosome binding sites, translation initiation andtermination codons, and elements derived from viral systems(particularly an internal ribosome entry site, or IRES, also referred toas a CITE sequence). The expression cassette may also include otherfeatures such as an origin of replication, and/or chromosome integrationelements such as retroviral long terminal repeats (LTRs), oradeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the bispecific antigen binding molecule or polypeptide fragmentsthereof is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding the bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal peptide or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Those of ordinary skill in theart are aware that polypeptides secreted by vertebrate cells generallyhave a signal peptide fused to the N-terminus of the polypeptide, whichis cleaved from the translated polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g. an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, may be used. Forexample, the wild-type leader sequence may be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling thefusion protein may be included within or at the ends of thepolynucleotide encoding a bispecific antigen binding molecule of theinvention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one or morepolynucleotides of the invention is provided. In certain aspects, a hostcell comprising one or more vectors of the invention is provided. Thepolynucleotides and vectors may incorporate any of the features, singlyor in combination, described herein in relation to polynucleotides andvectors, respectively. In one aspect, a host cell comprises (e.g. hasbeen transformed or transfected with) a vector comprising apolynucleotide that encodes (part of) a bispecific antigen bindingmolecule of the invention of the invention. As used herein, the term“host cell” refers to any kind of cellular system which can beengineered to generate the fusion proteins of the invention or fragmentsthereof. Host cells suitable for replicating and for supportingexpression of antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antigen binding molecule for clinicalapplications. Suitable host cells include prokaryotic microorganisms,such as E. coli, or various eukaryotic cells, such as Chinese hamsterovary cells (CHO), insect cells, or the like. For example, polypeptidesmay be produced in bacteria in particular when glycosylation is notneeded. After expression, the polypeptide may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004),and Li et al., Nat Biotech 24, 210-215 (2006).

Suitable host cells for the expression of (glycosylated) polypeptidesare also derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g., inGraham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad SciUSA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 andSp2/0. For a review of certain mammalian host cell lines suitable forprotein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, preferably a mammaliancell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonickidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an immunoglobulin, may be engineered so asto also express the other of the immunoglobulin chains such that theexpressed product is an immunoglobulin that has both a heavy and a lightchain.

In one aspect, a method of producing a bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof is provided,wherein the method comprises culturing a host cell comprisingpolynucleotides encoding the bispecific antigen binding molecule of theinvention or polypeptide fragments thereof, as provided herein, underconditions suitable for expression of the bispecific antigen bindingmolecule of the invention or polypeptide fragments thereof, andrecovering the bispecific antigen binding molecule of the invention orpolypeptide fragments thereof from the host cell (or host cell culturemedium).

Bispecific molecules of the invention prepared as described herein maybe purified by art-known techniques such as high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art. Foraffinity chromatography purification an antibody, ligand, receptor orantigen can be used to which the bispecific antigen binding moleculebinds. For example, for affinity chromatography purification of fusionproteins of the invention, a matrix with protein A or protein G may beused. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate an antigen bindingmolecule essentially as described in the examples. The purity of thebispecific antigen binding molecule or fragments thereof can bedetermined by any of a variety of well-known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like. For example, the bispecific antigen binding moleculesexpressed as described in the Examples were shown to be intact andproperly assembled as demonstrated by reducing and non-reducingSDS-PAGE.

Assays

The antigen binding molecules provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Affinity Assays

The affinity of the bispecific antigen binding molecules, antibodies andantibody fragments provided herein for the corresponding TNF receptorcan be determined in accordance with the methods set forth in theexamples by surface plasmon resonance (SPR), using standardinstrumentation such as a BIAcore instrument (GE Healthcare), andreceptors or target proteins such as may be obtained by recombinantexpression. The affinity of the bispecific antigen binding molecule forthe target cell antigen can also be determined by surface plasmonresonance (SPR), using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. A specific illustrative andexemplary embodiment for measuring binding affinity is described inExample 2. According to one aspect, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

2. Binding Assays and Other Assays

Binding of the bispecific antigen binding molecule provided herein tothe corresponding receptor expressing cells may be evaluated using celllines expressing the particular receptor or target antigen, for exampleby flow cytometry (FACS). In one aspect, peripheral blood mononuclearcells (PBMCs) expressing the TNF receptor are used in the binding assay.These cells are used directly after isolation (naïve PMBCs) or afterstimulation (activated PMBCs). In another aspect, activated mousesplenocytes (expressing the TNF receptor molecule) were used todemonstrate the binding of the bispecific antigen binding molecule orantibody of the invention to the corresponding TNF receptor expressingcells. In a further aspect, PBMC isolated from heparinized blood ofhealthy Macaca fascicularis were used to show of the bispecific antigenbinding molecule or antibody to the corresponding cynomolgus TNFreceptor expressing cells.

In a further aspect, cancer cell lines expressing the target cellantigen, for example FAP, were used to demonstrate the binding of theantigen binding molecules to the target cell antigen.

In another aspect, competition assays may be used to identify an antigenbinding molecule that competes with a specific antibody or antigenbinding molecule for binding to the target or TNF receptor,respectively. In certain embodiments, such a competing antigen bindingmolecule binds to the same epitope (e.g., a linear or a conformationalepitope) that is bound by a specific anti-target antibody or a specificanti-TNF receptor antibody. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.).

3. Activity Assays

In one aspect, assays are provided for identifying bispecific antigenbinding molecules that bind to a specific target cell antigen and to aspecific TNF receptor having biological activity. Biological activitymay include, e.g., agonistic signalling through the TNF receptor oncells expressing the target cell antigen. TNF family ligandtrimer-containing antigen binding molecules identified by the assays ashaving such biological activity in vitro are also provided.

In certain aspects, a bispecific antigen binding molecule of theinvention is tested for such biological activity. Furthermore, assaysfor detecting cell lysis (e.g. by measurement of LDH release), inducedapoptosis kinetics (e.g. by measurement of Caspase 3/7 activity) orapoptosis (e.g. using the TUNEL assay) are well known in the art. Inaddition the biological activity of such complexes can be assessed byevaluating their effects on survival, proliferation and lymphokinesecretion of various lymphocyte subsets such as NK cells, NKT-cells orγδ T-cells or assessing their capacity to modulate phenotype andfunction of antigen presenting cells such as dendritic cells,monocytes/macrophages or B-cells.

Pharmaceutical Compositions, Formulations and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the bispecific antigen binding molecules or antibodiesprovided herein, e.g., for use in any of the below therapeutic methods.In one embodiment, a pharmaceutical composition comprises any of thebispecific antigen binding molecules provided herein and at least onepharmaceutically acceptable excipient. In another embodiment, apharmaceutical composition comprises any of the bispecific antigenbinding molecules provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antigenbinding molecules dissolved or dispersed in a pharmaceuticallyacceptable excipient. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e. do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one bispecific antigen binding molecule or antibodyaccording to the invention and optionally an additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilized formulationsor aqueous solutions. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, buffers, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g. antibacterialagents, antifungal agents), isotonic agents, salts, stabilizers andcombinations thereof, as would be known to one of ordinary skill in theart.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the bispecific antigen binding molecules or antibodies ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the bispecific antigen binding molecules orantibodies may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the antigen bindingmolecules of the invention in the required amount in the appropriatesolvent with various of the other ingredients enumerated below, asrequired. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and/or theother ingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable excipients include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

In addition to the compositions described previously, the antigenbinding molecules may also be formulated as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the fusion proteins may be formulated withsuitable polymeric or hydrophobic materials (for example as emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the bispecific antigen bindingmolecules or antibodies of the invention may be manufactured by means ofconventional mixing, dissolving, emulsifying, encapsulating, entrappingor lyophilizing processes. Pharmaceutical compositions may be formulatedin conventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The bispecific antigen binding molecules may be formulated into acomposition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g. those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Pharmaceutical salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free base forms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the bispecific antigen binding molecules or antibodies providedherein may be used in therapeutic methods.

For use in therapeutic methods, bispecific antigen binding molecules orantibodies of the invention can be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, bispecific antigen binding molecules or antibodies of theinvention for use as a medicament are provided.

In further aspects, bispecific antigen binding molecules or antibodiesof the invention for use (i) in stimulating or enhancing T cellresponse, (ii) for use in supporting survival of activated T cells,(iii) for use in the treatment of infections, (iv) for use in thetreatment of cancer, (v) for use in delaying progression of cancer, or(vi) for use in prolonging the survival of a patient suffering fromcancer, are provided. In a particular aspect, TNF family ligandtrimer-containing antigen binding molecules or antibodies of theinvention for use in treating a disease, in particular for use in thetreatment of cancer, are provided.

In certain aspects, bispecific antigen binding molecules or antibodiesof the invention for use in a method of treatment are provided. In oneaspect, the invention provides a bispecific antigen binding molecule orantibody as described herein for use in the treatment of a disease in anindividual in need thereof. In certain aspects, the invention provides abispecific antigen binding molecule or antibody for use in a method oftreating an individual having a disease comprising administering to theindividual a therapeutically effective amount of the bispecific antigenbinding molecule or antibody. In certain aspects the disease to betreated is cancer. The subject, patient, or “individual” in need oftreatment is typically a mammal, more specifically a human.

In one aspect, provided is a method for (i) stimulating or enhancingT-cell response, (ii) supporting survival of activated T cells, (iii)treating infections, (iv) treating cancer, (v) delaying progression ofcancer or (vi) prolonging the survival of a patient suffering fromcancer, wherein the method comprises administering a therapeuticallyeffective amount of the bispecific antigen binding molecule or antibodyof the invention to an individual in need thereof.

In a further aspect, the invention provides for the use of thebispecific antigen binding molecule or antibody of the invention in themanufacture or preparation of a medicament for the treatment of adisease in an individual in need thereof. In one aspect the medicamentis for use in a method of treating a disease comprising administering toan individual having the disease a therapeutically effective amount ofthe medicament. In certain aspects, the disease to be treated is aproliferative disorder, particularly cancer. Examples of cancersinclude, but are not limited to, bladder cancer, brain cancer, head andneck cancer, pancreatic cancer, lung cancer, breast cancer, ovariancancer, uterine cancer, cervical cancer, endometrial cancer, esophagealcancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer,prostate cancer, blood cancer, skin cancer, squamous cell carcinoma,bone cancer, and kidney cancer. Other examples of cancer includecarcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma),blastoma, sarcoma, and leukemia. Other cell proliferation disorders thatcan be treated using the bispecific antigen binding molecule or antibodyof the invention include, but are not limited to neoplasms located inthe: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system. Also included arepre-cancerous conditions or lesions and cancer metastases. In certainembodiments the cancer is chosen from the group consisting of renal cellcancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. A skilled artisan readily recognizesthat in many cases the bispecific antigen binding molecule or antibodyof the invention may not provide a cure but may provide a benefit. Insome aspects, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some aspects, an amountof the bispecific antigen binding molecule or antibody of the inventionthat provides a physiological change is considered an “effective amount”or a “therapeutically effective amount”.

For the prevention or treatment of disease, the appropriate dosage of abispecific antigen binding molecule or antibody of the invention (whenused alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, thespecific molecule, the severity and course of the disease, whether thebispecific antigen binding molecule or antibody of the invention isadministered for preventive or therapeutic purposes, previous orconcurrent therapeutic interventions, the patient's clinical history andresponse to the fusion protein, and the discretion of the attendingphysician. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

The bispecific antigen binding molecule or antibody of the invention issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of TNF family ligandtrimer-containing antigen binding molecule can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentwould generally be sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the bispecific antigen bindingmolecule or antibody of the invention would be in the range from about0.005 mg/kg to about 10 mg/kg. In other examples, a dose may alsocomprise from about 1 μg/kg body weight, about 5 μg/kg body weight,about 10 μg/kg body weight, about 50 μg/kg body weight, about 100 μg/kgbody weight, about 200 μg/kg body weight, about 350 μg/kg body weight,about 500 μg/kg body weight, about 1 mg/kg body weight, about 5 mg/kgbody weight, about 10 mg/kg body weight, about 50 mg/kg body weight,about 100 mg/kg body weight, about 200 mg/kg body weight, about 350mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kg bodyweight or more per administration, and any range derivable therein. Inexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg body weight to about 100 mg/kg body weight, about 5 μg/kgbody weight to about 500 mg/kg body weight etc., can be administered,based on the numbers described above. Thus, one or more doses of about0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof)may be administered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the fusion protein). An initial higher loading dose, followed by oneor more lower doses may be administered. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The bispecific antigen binding molecule or antibody of the inventionwill generally be used in an amount effective to achieve the intendedpurpose. For use to treat or prevent a disease condition, the bispecificantigen binding molecule or antibody of the invention, or pharmaceuticalcompositions thereof, are administered or applied in a therapeuticallyeffective amount. Determination of a therapeutically effective amount iswell within the capabilities of those skilled in the art, especially inlight of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the bispecific antigen binding molecule or antibody ofthe invention which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the bispecific antigen binding molecule orantibody of the invention may not be related to plasma concentration.One skilled in the art will be able to optimize therapeuticallyeffective local dosages without undue experimentation.

A therapeutically effective dose of the bispecific antigen bindingmolecule or antibody of the invention described herein will generallyprovide therapeutic benefit without causing substantial toxicity.Toxicity and therapeutic efficacy of a fusion protein can be determinedby standard pharmaceutical procedures in cell culture or experimentalanimals. Cell culture assays and animal studies can be used to determinethe LD₅₀ (the dose lethal to 50% of a population) and the ED₅₀ (the dosetherapeutically effective in 50% of a population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, whichcan be expressed as the ratio LD₅₀/ED₅₀. Bispecific antigen bindingmolecules that exhibit large therapeutic indices are preferred. In oneaspect, the bispecific antigen binding molecule or antibody of theinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with fusion proteins of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The bispecific antigen binding molecule or antibody of the invention maybe administered in combination with one or more other agents in therapy.For instance, the bispecific antigen binding molecule or antibody of theinvention of the invention may be co-administered with at least oneadditional therapeutic agent. The term “therapeutic agent” encompassesany agent that can be administered for treating a symptom or disease inan individual in need of such treatment. Such additional therapeuticagent may comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is another anti-cancer agent, for example amicrotubule disruptor, an antimetabolite, a topoisomerase inhibitor, aDNA intercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent. In certain aspects, an additionaltherapeutic agent is an immunomodulatory agent, a cytostatic agent, aninhibitor of cell adhesion, a cytotoxic or cytostatic agent, anactivator of cell apoptosis, or an agent that increases the sensitivityof cells to apoptotic inducers.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The bispecificantigen binding molecule or antibody of the invention are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the bispecific antigen binding molecule or antibody ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific antigen binding molecule or antibodyof the invention.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises a bispecific antigen binding moleculeof the invention; and (b) a second container with a compositioncontained therein, wherein the composition comprises a further cytotoxicor otherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C (Sequences): SEQ ID NO: Name Sequence 1 Human OX40 ECD UniprotNo. P43489, aa 29-214 2 OX40(8H9, 49B4, 1G4, SYAIS 20B7) CDR-H1 3OX40(CLC-563, CLC- SYAMS 564, 17A9) CDR-H1 4 OX40(8H9, 49B4, 1G4,GIIPIFGTANYAQKFQG 20B7) CDR-H2 5 OX40(CLC-563, CLC- AISGSGGSTYYADSVKG564, 17A9) CDR-H2 6 OX40(8H9) CDR-H3 EYGWMDY 7 OX40(49B4) CDR-H3EYYRGPYDY 8 OX40(1G4) CDR-H3 EYGSMDY 9 OX40(20B7) CDR-H3 VNYPYSYWGDFDY10 OX40(CLC-563) CDR-H3 DVGAFDY 11 OX40(CLC-564) CDR-H3 DVGPFDY 12OX40(17A9)-CDR-H3 VFYRGGVSMDY 13 OX40(8H9, 49B4, 1G4, RASQSISSWLA 20B7)CDR-L1 14 OX40(CLC-563, CLC564) RASQSVSSSYLA CDR-L1 15 OX40(17A9) CDR-L1QGDSLRSYYAS 16 OX40(8H9, 49B4, 1G4, DASSLES 20B7) CDR-L2 17OX40(CLC-563, CLC564) GASSRAT CDR-L2 18 OX40(17A9) CDR-L2 GKNNRPS 19OX40(8H9) CDR-L3 QQYLTYSRFT 20 OX40(49B4) CDR-L3 QQYSSQPYT 21 OX40(1G4)CDR-L3 QQYISYSMLT 22 OX40(20B7) CDR-L3 QQYQAFSLT 23 OX40(CLC-563, CLC-QQYGSSPLT 164) CDR-L3 24 OX40(17A9) CDR-L3 NSRVMPHNRV 25 OX40(8H9) VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA DKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSS 26 OX40(8H9) VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIK27 OX40(49B4) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA DKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSS 28 OX40(49B4) VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYSSQPYTFGQGTKVEIK29 OX40(1G4) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA DKSTSTAYMELSSLRSEDTAVYYCAREYGSMDYWGQGTTVTVSS 30 OX40(1G4) VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYISYSMLTFGQGTKVEIK31 OX40(20B7) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA DKSTSTAYMELSSLRSEDTAVYYCARVNYPYSYWGDFDYWGQGTTVTVSS 32 OX40(20B7) VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYQAFSLTFGQGTKVEIK33 OX40(CLC-563) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSS 34 OX40(CLC-563) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK35 OX40(CLC-564) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAFDVGPFDYWGQGTLVTVSS 36 OX40(CLC-564) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK37 OX40(17A9) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARVFYRGGVSMDYWGQGTLVTVSS 38 OX40(17A9) VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTI TGAQAEDEADYYCNSRVMPHNRVFGGGTKLTV39 Human 4-1BB ECD Uniprot No. Q07011, aa 24-186 40 4-1BB(12B3, 11D5,9B11, SYAIS 20G2) CDR-H1 41 4-1BB(25G7) CDR-H1 SYAMS 42 4-1BB(12B3,11D5, 9B11, GIIPIFGTANYAQKFQG 20G2) CDR-H2 43 4-1BB(25G7) CDR-H2AISGSGGSTYYADSVKG 44 4-1BB(12B3) CDR-H3 SEFRFYADFDY 45 4-1BB(25G7)CDR-H3 DDPWPPFDY 46 4-1BB(11D5) CDR-H3 STLIYGYFDY 47 4-1BB(9B11) CDR-H3SSGAYPGYFDY 48 4-1BB(20G2) CDR-H3 SYYWESYPFDY 49 4-1BB(12B3, 11D5, 9B11,RASQSISSWLA 20G2) CDR-L1 50 4-1BB(25G7) CDR-L1 QGDSLRSYYAS 514-1BB(12B3, 11D5, 9B11, DASSLES 20G2) CDR-L2 52 4-1BB(25G7) CDR-L2GKNNRPS 53 4-1BB(12B3) CDR-L3 QQYHSYPQT 54 4-1BB (25G7) CDR-L3NSLDRRGMWV 55 4-1BB(11D5) CDR-L3 QQLNSYPQT 56 4-1BB(9B11) CDR-L3QQVNSYPQT 57 4-1BB(20G2) CDR-L3 QQQHSYYT 58 4-1BB(12B3) VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSEFRFYADFD YWGQGTTVTVSS 59 4-1BB(12B3) VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQYHSYPQTFGQGTKVEIK60 4-1BB(25G7) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSS 61 4-1BB(25G7) VL SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTI TGAQAEDEADYYCNSLDRRGMWVFGGGTKLTV62 4-1BB(11D5) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTLIYGYFDY WGQGTTVTVSS 63 4-1BB(11D5) VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQLNSYPQTFGQGTKVEIK64 4-1BB(9B11) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSSGAYPGYFD YWGQGTTVTVSS 65 4-1BB(9B11) VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQVNSYPQTFGQGTKVEIK66 4-1BB(20G2) VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYYWESYPFD YWGQGTTVTVSS 67 4-1BB(20G2) VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTI SSLQPDDFATYYCQQQHSYYTFGQGTKVEIK68 FAP(28H1) CDR-H1 SHAMS 69 FAP(4B9) CDR-H1 SYAMS 70 FAP(28H1) CDR-H2AIWASGEQYYADSVKG 71 FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 72 FAP(28H1)CDR-H3 GWLGNFDY 73 FAP(4B9) CDR-H3 GWFGGFNY 74 FAP(28H1) CDR-L1RASQSVSRSYLA 75 FAP(4B9) CDR-L1 RASQSVTSSYLA 76 FAP(28H1) CDR-L2 GASTRAT77 FAP(4B9) CDR-L2 VGSRRAT 78 FAP(28H1) CDR-L3 QQGQVIPPT 79 FAP(4B9)CDR-L3 QQGIMLPPT 80 FAP(28H1) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS 81 FAP(28H1) VL EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIK82 FAP(4B9) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRD NSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 83 FAP(4B9) VL EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQGIMLPPTFGQGTKVEIK84 Human (hu) FAP UniProt no. Q12884 85 hu FAP ectodomain + poly-RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS lys-tag + his₆-tagGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAE YFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFS LSDGKKKKKKGHHHHHH 86 nucleotidesequence CGCCCTTCAAGAGTTCATAACTCTGAAGAAAATAC hu FAP ectodomain + poly-AATGAGAGCACTCACACTGAAGGATATTTTAAATG lys-tag + his₆-tagGAACATTTTCTTATAAAACATTTTTTCCAAACTGGA TTTCAGGACAAGAATATCTTCATCAATCTGCAGATAACAATATAGTACTTTATAATATTGAAACAGGACA ATCATATACCATTTTGAGTAATAGAACCATGAAAAGTGTGAATGCTTCAAATTACGGCTTATCACCTGAT CGGCAATTTGTATATCTAGAAAGTGATTATTCAAAGCTTTGGAGATACTCTTACACAGCAACATATTACA TCTATGACCTTAGCAATGGAGAATTTGTAAGAGGAAATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGG TCGCCTGTTGGGAGTAAATTAGCATATGTCTATCAAAACAATATCTATTTGAAACAAAGACCAGGAGAT CCACCTTTTCAAATAACATTTAATGGAAGAGAAAATAAAATATTTAATGGAATCCCAGACTGGGTTTATG AAGAGGAAATGCTTGCTACAAAATATGCTCTCTGGTGGTCTCCTAATGGAAAATTTTTGGCATATGCGGA ATTTAATGATACGGATATACCAGTTATTGCCTATTCCTATTATGGCGATGAACAATATCCTAGAACAATAA ATATTCCATACCCAAAGGCTGGAGCTAAGAATCCCGTTGTTCGGATATTTATTATCGATACCACTTACCCT GCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGCAATGATAGCCTCAAGTGATTATTATTTCAGTTGGC TCACGTGGGTTACTGATGAACGAGTATGTTTGCAGTGGCTAAAAAGAGTCCAGAATGTTTCGGTCCTGTC TATATGTGACTTCAGGGAAGACTGGCAGACATGGGATTGTCCAAAGACCCAGGAGCATATAGAAGAAAG CAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAACACCAGTTTTCAGCTATGATGCCATTTCGTACTACA AAATATTTAGTGACAAGGATGGCTACAAACATATTCACTATATCAAAGACACTGTGGAAAATGCTATTCA AATTACAAGTGGCAAGTGGGAGGCCATAAATATATTCAGAGTAACACAGGATTCACTGTTTTATTCTAG CAATGAATTTGAAGAATACCCTGGAAGAAGAAACATCTACAGAATTAGCATTGGAAGCTATCCTCCAAG CAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAAGGTGCCAATATTACACAGCAAGTTTCAGCGACTAC GCCAAGTACTATGCACTTGTCTGCTACGGCCCAGGCATCCCCATTTCCACCCTTCATGATGGACGCACTG ATCAAGAAATTAAAATCCTGGAAGAAAACAAGGAATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTA AAGAGGAAATTAAGAAACTTGAAGTAGATGAAATTACTTTATGGTACAAGATGATTCTTCCTCCTCAATT TGACAGATCAAAGAAGTATCCCTTGCTAATTCAAGTGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCT GTATTTGCTGTTAATTGGATATCTTATCTTGCAAGTAAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCG AGGAACAGCTTTCCAAGGTGACAAACTCCTCTATGCAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAA GACCAGATTACAGCTGTCAGAAAATTCATAGAAATGGGTTTCATTGATGAAAAAAGAATAGCCATATGGG GCTGGTCCTATGGAGGATACGTTTCATCACTGGCCCTTGCATCTGGAACTGGTCTTTTCAAATGTGGTATA GCAGTGGCTCCAGTCTCCAGCTGGGAATATTACGCGTCTGTCTACACAGAGAGATTCATGGGTCTCCCAA CAAAGGATGATAATCTTGAGCACTATAAGAATTCAACTGTGATGGCAAGAGCAGAATATTTCAGAAATGT AGACTATCTTCTCATCCACGGAACAGCAGATGATAATGTGCACTTTCAAAACTCAGCACAGATTGCTAAA GCTCTGGTTAATGCACAAGTGGATTTCCAGGCAATGTGGTACTCTGACCAGAACCACGGCTTATCCGGCC TGTCCACGAACCACTTATACACCCACATGACCCACTTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGCAA AAAGAAAAAGAAAAAGGGCCACCACCATCACCATCAC 87 mouse FAP UniProt no. P97321 88 Murine FAPRPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWIS ectodomain + poly-lys-EQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNAT tag + his₆-tagDYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQNGEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKYALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPRTINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVPEMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVL SICDFREDWHAWECPKNQEHVEESRTGWAGGFFVSTPAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIGNSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVCYGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLPKVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTAFQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFIDEERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARA EYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCF SLSDGKKKKKKGHHHHHH 89 nucleotidesequence CGTCCCTCAAGAGTTTACAAACCTGAAGGAAACAC Murine FAPAAAGAGAGCTCTTACCTTGAAGGATATTTTAAATG ectodomain + poly-lys-GAACATTCTCATATAAAACATATTTTCCCAACTGG tag + his₆-tagATTTCAGAACAAGAATATCTTCATCAATCTGAGGA TGATAACATAGTATTTTATAATATTGAAACAAGAGAATCATATATCATTTTGAGTAATAGCACCATGAAA AGTGTGAATGCTACAGATTATGGTTTGTCACCTGATCGGCAATTTGTGTATCTAGAAAGTGATTATTCAA AGCTCTGGCGATATTCATACACAGCGACATACTACATCTACGACCTTCAGAATGGGGAATTTGTAAGAGG ATACGAGCTCCCTCGTCCAATTCAGTATCTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTATAT CAAAACAATATTTATTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACTTATACTGGAAGAGAA AATAGAATATTTAATGGAATACCAGACTGGGTTTATGAAGAGGAAATGCTTGCCACAAAATATGCTCTTT GGTGGTCTCCAGATGGAAAATTTTTGGCATATGTAGAATTTAATGATTCAGATATACCAATTATTGCCTA TTCTTATTATGGTGATGGACAGTATCCTAGAACTATAAATATTCCATATCCAAAGGCTGGGGCTAAGAAT CCGGTTGTTCGTGTTTTTATTGTTGACACCACCTACCCTCACCACGTGGGCCCAATGGAAGTGCCAGTTCC AGAAATGATAGCCTCAAGTGACTATTATTTCAGCTGGCTCACATGGGTGTCCAGTGAACGAGTATGCTTG CAGTGGCTAAAAAGAGTGCAGAATGTCTCAGTCCTGTCTATATGTGATTTCAGGGAAGACTGGCATGCAT GGGAATGTCCAAAGAACCAGGAGCATGTAGAAGAAAGCAGAACAGGATGGGCTGGTGGATTCTTTGTTT CGACACCAGCTTTTAGCCAGGATGCCACTTCTTACTACAAAATATTTAGCGACAAGGATGGTTACAAACA TATTCACTACATCAAAGACACTGTGGAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATATAT ATATTCCGCGTAACACAGGATTCACTGTTTTATTCTAGCAATGAATTTGAAGGTTACCCTGGAAGAAGAA ACATCTACAGAATTAGCATTGGAAACTCTCCTCCGAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGA AAGGTGCCAATATTACACAGCAAGTTTCAGCTACAAAGCCAAGTACTATGCACTCGTCTGCTATGGCCCT GGCCTCCCCATTTCCACCCTCCATGATGGCCGCACAGACCAAGAAATACAAGTATTAGAAGAAAACAAA GAACTGGAAAATTCTCTGAGAAATATCCAGCTGCCTAAAGTGGAGATTAAGAAGCTCAAAGACGGGGGA CTGACTTTCTGGTACAAGATGATTCTGCCTCCTCAGTTTGACAGATCAAAGAAGTACCCTTTGCTAATTCA AGTGTATGGTGGTCCTTGTAGCCAGAGTGTTAAGTCTGTGTTTGCTGTTAATTGGATAACTTATCTCGCAA GTAAGGAGGGGATAGTCATTGCCCTGGTAGATGGTCGGGGCACTGCTTTCCAAGGTGACAAATTCCTGCA TGCCGTGTATCGAAAACTGGGTGTATATGAAGTTGAGGACCAGCTCACAGCTGTCAGAAAATTCATAGA AATGGGTTTCATTGATGAAGAAAGAATAGCCATATGGGGCTGGTCCTACGGAGGTTATGTTTCATCCCTG GCCCTTGCATCTGGAACTGGTCTTTTCAAATGTGGCATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATT ACGCATCTATCTACTCAGAGAGATTCATGGGCCTCCCAACAAAGGACGACAATCTCGAACACTATAAAA ATTCAACTGTGATGGCAAGAGCAGAATATTTCAGAAATGTAGACTATCTTCTCATCCACGGAACAGCAGA TGATAATGTGCACTTTCAGAACTCAGCACAGATTGCTAAAGCTTTGGTTAATGCACAAGTGGATTTCCAG GCGATGTGGTACTCTGACCAGAACCATGGTATATTATCTGGGCGCTCCCAGAATCATTTATATACCCACA TGACGCACTTCCTCAAGCAATGCTTTTCTTTATCAGACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCA TCACCATCAC 90 Cynomolgus FAPRPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS ectodomain + poly-lys-GQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNA tag + his₆-tagSNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKYALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAP VSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNA QVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDGKKKKKKGHHHHHH 91 nucleotide sequenceCGCCCTCCAAGAGTTCATAACTCTGAAGAAAATAC Cynomolgus FAPAATGAGAGCACTCACACTGAAGGATATTTTAAATG ectodomain + poly-lys-GGACATTTTCTTATAAAACATTTTTTCCAAACTGGA tag + his₆-tagTTTCAGGACAAGAATATCTTCATCAATCTGCAGAT AACAATATAGTACTTTATAATATTGAAACAGGACAATCATATACCATTTTGAGTAACAGAACCATGAAAA GTGTGAATGCTTCAAATTATGGCTTATCACCTGATCGGCAATTTGTATATCTAGAAAGTGATTATTCAAA GCTTTGGAGATACTCTTACACAGCAACATATTACATCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTCTATCA AAACAATATCTATTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACATTTAATGGAAGAGAAAA TAAAATATTTAATGGAATCCCAGACTGGGTTTATGAAGAGGAAATGCTTGCTACAAAATATGCTCTCTGG TGGTCTCCTAATGGAAAATTTTTGGCATATGCGGAATTTAATGATACAGATATACCAGTTATTGCCTATTC CTATTATGGCGATGAACAATATCCCAGAACAATAAATATTCCATACCCAAAGGCCGGAGCTAAGAATCCT TTTGTTCGGATATTTATTATCGATACCACTTACCCTGCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGC AATGATAGCCTCAAGTGATTATTATTTCAGTTGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAG TGGCTAAAAAGAGTCCAGAATGTTTCGGTCTTGTCTATATGTGATTTCAGGGAAGACTGGCAGACATGGG ATTGTCCAAAGACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAA CACCAGTTTTCAGCTATGATGCCATTTCATACTACAAAATATTTAGTGACAAGGATGGCTACAAACATATT CACTATATCAAAGACACTGTGGAAAATGCTATTCAAATTACAAGTGGCAAGTGGGAGGCCATAAATATA TTCAGAGTAACACAGGATTCACTGTTTTATTCTAGCAATGAATTTGAAGATTACCCTGGAAGAAGAAAC ATCTACAGAATTAGCATTGGAAGCTATCCTCCAAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAA GGTGCCAATATTACACAGCAAGTTTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTATGGCCCAGG CATCCCCATTTCCACCCTTCATGACGGACGCACTGATCAAGAAATTAAAATCCTGGAAGAAAACAAGGA ATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTAAAGAGGAAATTAAGAAACTTGAAGTAGATGAAAT TACTTTATGGTACAAGATGATTCTTCCTCCTCAATTTGACAGATCAAAGAAGTATCCCTTGCTAATTCAAG TGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCTGTATTTGCTGTTAATTGGATATCTTATCTTGCAAGT AAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCGGGGAACAGCTTTCCAAGGTGACAAACTCCTGTATG CAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAAGACCAGATTACAGCTGTCAGAAAATTCATAGAAAT GGGTTTCATTGATGAAAAAAGAATAGCCATATGGGGCTGGTCCTATGGAGGATATGTTTCATCACTGGCC CTTGCATCTGGAACTGGTCTTTTCAAATGTGGGATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATTACG CGTCTGTCTACACAGAGAGATTCATGGGTCTCCCAACAAAGGATGATAATCTTGAGCACTATAAGAATTC AACTGTGATGGCAAGAGCAGAATATTTCAGAAATGTAGACTATCTTCTCATCCACGGAACAGCAGATGA TAATGTGCACTTTCAAAACTCAGCACAGATTGCTAAAGCTCTGGTTAATGCACAAGTGGATTTCCAGGCA ATGTGGTACTCTGACCAGAACCACGGCTTATCCGGCCTGTCCACGAACCACTTATACACCCACATGACCC ACTTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCATCACC ATCAC 92 human CEA UniProt no. P0673193 human MCSP UniProt no. Q6UVK1 94 human EGFR UniProt no. P00533 95human CD19 UniProt no. P15391 96 human CD20 Uniprot no. P11836 97 humanCD33 UniProt no. P20138 98 human OX40 UniProt no. P43489 99 human 4-1BBUniProt no. Q07011 100 human CD27 UniProt no. P26842 101 human HVEMUniProt no. Q92956 102 human CD30 UniProt no. P28908 103 human GITRUniProt no. Q9Y5U5 104 murine OX40 UniProt no. P47741 105 murine 4-1BBUniProt no. P20334 106 cynomolgus 4-1BB Uniprot no. F6W5G6 107 Peptidelinker (G4S) GGGGS 108 Peptide linker (G4S)₂ GGGGSGGGGS 109 Peptidelinker (SG4)₂ SGGGGSGGGG 110 Peptide linker G4(SG4)₂ GGGGSGGGGSGGGG 111Peptide linker GSPGSSSSGS 112 Peptide linker (G4S)₃ GGGGSGGGGSGGGGS 113Peptide linker (G4S)₄ GGGGSGGGGSGGGGSGGGGS 114 Peptide linker GSGSGSGS115 Peptide linker GSGSGNGS 116 Peptide linker GGSGSGSG 117 Peptidelinker GGSGSG 118 Peptide linker GGSG 119 Peptide linker GGSGNGSG 120Peptide linker GGNGSGSG 121 Peptide linker GGNGSG 122 cynomolgus OX40ECD aa 29-214 123 murine OX40 ECD aa 10-211 124 Nucleotide sequence seeTable 2 Fc hole chain 125 Nucleotide sequence see Table 2 human OX40antigen Fc knob chain 126 Nucleotide sequence see Table 2 cynomolgusOX40 antigen Fc knob chain 127 Nucleotide sequence see Table 2 murineOX40 antigen Fc knob chain 128 Fc hole chain see Table 2 129 human OX40antigen Fc see Table 2 knob chain 130 cynomolgus OX40 antigen see Table2 Fc knob chain 131 murine OX40 antigen Fc see Table 2 knob chain 132nucleotide sequence of see Table 3 library DP88-4 133 nucleotidesequence of see Table 4 Fab light chain Vk1_5 134 Fab light chain Vk1_5see Table 4 135 nucleotide sequence of see Table 4 Fab heavy chainVH1_69 136 Fab heavy chain VH1_69 see Table 4 137 LMB3 see Table 5 138Vk1_5_L3r_S see Table 5 139 Vk1_5_L3r_SY see Table 5 140 Vk1_5_L3r_SPYsee Table 5 141 RJH31 see Table 5 142 RJH32 see Table 5 143 DP88-v4-4see Table 5 144 DP88-v4-6 see Table 5 145 DP88-v4-8 see Table 5 146fdseqlong see Table 5 147 (Vk3_20/VH3_23) see Table 6 template 148nucleotide sequence of see Table 7 Fab light chain Vk3_20 149 Fab lightchain Vk3_20 see Table 7 150 nucleotide sequence of see Table 7 Fabheavy chain VH3_23 151 Fab heavy chain VH3_23 see Table 7 (DP47) 152MS64 see Table 8 153 DP47CDR3_ba (mod.) see Table 8 154 DP47-v4-4 seeTable 8 155 DP47-v4-6 see Table 8 156 DP47-v4-8 see Table 8 157fdseqlong see Table 8 158 Vl3_19/VH3_23 library see Table 9 template 159nucleotide sequence of see Table 10 Fab light chain Vl3_19 160 Fab lightchain Vl3_19 see Table 10 161 LMB3 see Table 11 162 Vl_3_19_L3r_V seeTable 11 163 Vl_3_19_L3r_HV see Table 11 164 Vl_3_19_L3r_HLV see Table11 165 RJH80 see Table 11 166 Nucleotide sequence see Table 12 OX40(8H9)VL 167 Nucleotide sequence see Table 12 OX40(8H9) VH 168 Nucleotidesequence see Table 12 OX40(49B4) VL 169 Nucleotide sequence see Table 12OX40(49B4) VH 170 Nucleotide sequence see Table 12 OX40(1G4) VL 171Nucleotide sequence see Table 12 OX40(1G4) VH 172 Nucleotide sequencesee Table 12 OX40(20B7) VL 173 Nucleotide sequence see Table 12OX40(20B7) VH 174 Nucleotide sequence see Table 12 OX40(CLC-563) VL 175Nucleotide sequence see Table 12 OX40(CLC-563) VH 176 Nucleotidesequence see Table 12 OX40(CLC-564) VL 177 Nucleotide sequence see Table12 OX40(CLC-564) VH 178 Nucleotide sequence see Table 12 OX40(17A9) VL179 Nucleotide sequence see Table 12 OX40(17A9) VH 180 8H9 P329GLALAIgG1 nucleotide sequence, see Table 13 (light chain) 181 8H9 P329GLALAIgG1 nucleotide sequence, see Table 13 (heavy chain) 182 8H9 P329GLALAIgG1 see Table 13 (light chain) 183 8H9 P329GLALA IgG1 see Table 13(heavy chain) 184 49B4 P329GLALA IgG1 nucleotide sequence, see Table 13(light chain) 185 49B4 P329GLALA IgG1 nucleotide sequence, see Table 13(heavy chain) 186 49B4 P329GLALA IgG1 see Table 13 (light chain) 18749B4 P329GLALA IgG1 see Table 13 (heavy chain) 188 1G4 P329GLALA IgG1nucleotide sequence, see Table 13 (light chain) 189 1G4 P329GLALA IgG1nucleotide sequence, see Table 13 (heavy chain) 190 1G4 P329GLALA IgG1see Table 13 (light chain) 191 1G4 P329GLALA IgG1 see Table 13 (heavychain) 192 20B7 P329GLALA IgG1 nucleotide sequence, see Table 13 (lightchain) 193 20B7 P329GLALA IgG1 nucleotide sequence, see Table 13 (heavychain) 194 20B7 P329GLALA IgG1 see Table 13 (light chain) 195 20B7P329GLALA IgG1 see Table 13 (heavy chain) 196 CLC-563 P329GLALAnucleotide sequence, see Table 13 IgG1 (light chain) 197 CLC-563P329GLALA nucleotide sequence, see Table 13 IgG1 (heavy chain) 198CLC-563 P329GLALA see Table 13 IgG1 (light chain) 199 CLC-563 P329GLALAsee Table 13 IgG1 (heavy chain) 200 CLC-564 P329GLALA nucleotidesequence, see Table 13 IgG1 (light chain) 201 CLC-564 P329GLALAnucleotide sequence, see Table 13 IgG1 (heavy chain) 202 CLC-564P329GLALA see Table 13 IgG1 (light chain) 203 CLC-564 P329GLALA seeTable 13 IgG1 (heavy chain) 204 17A9 P329GLALA IgG1 nucleotide sequence,see Table 13 (light chain) 205 17A9 P329GLALA IgG1 nucleotide sequence,see Table 13 (heavy chain) 206 17A9 P329GLALA IgG1 see Table 13 (lightchain) 207 17A9 P329GLALA IgG1 see Table 13 (heavy chain) 208 human OX40His nucleotide sequence 209 human OX40 His see Table 15 210 murine OX40His nucleotide sequence 211 murine OX40 His see Table 15 212 Nucleotidesequence of see Table 21 dimeric human OX40 antigen Fc 213 dimeric humanOX40 see Table 21 antigen Fc 214 (8B9) VHCH1-Heavy nucleotide sequence,see Table 25 chain-(28H1) VHCL 215 VLCH1-Light chain 2 nucleotidesequence, see Table 25 (28H1) 216 (8B9) VHCH1-Heavy see Table 25chain-(28H1) VHCL 217 VLCH1-Light chain 2 see Table 25 (28H1) 218 (49B4)VHCH1-Heavy nucleotide sequence, see Table 25 chain-(28H1) VHCL 219(49B4) VHCH1-Heavy see Table 25 chain-(28H1) VHCL 220 (1G4) VHCH1-Heavynucleotide sequence, see Table 25 chain-(28H1) VHCL 221 (1G4)VHCH1-Heavy see Table 25 chain-(28H1) VHCL 222 (20B7) VHCH1-Heavynucleotide sequence, see Table 25 chain-(28H1) VHCL 223 (20B7)VHCH1-Heavy see Table 25 chain-(28H1) VHCL 224 (CLC-563) VHCH1-Heavynucleotide sequence, see Table 25 chain-(28H1) VHCL 225 (CLC-563)VHCH1-Heavy see Table 25 chain-(28H1) VHCL 226 (CLC-564) VHCH1-Heavynucleotide sequence, see Table 25 chain-(28H1) VHCL 227 (CLC-564)VHCH1-Heavy see Table 25 chain-(28H1) VHCL 228 (28H1) VHCL-heavy chainnucleotide sequence, see Table 27 hole 229 (28H1) VHCL-heavy chain seeTable 27 hole 230 (49B4) VHCH1-heavy nucleotide sequence, see Table 27chain knob 231 (49B4) VHCH1-heavy see Table 27 chain knob 232 (1G4)VHCH1-heavy nucleotide sequence, see Table 27 chain knob 233 (1G4)VHCH1-heavy see Table 27 chain knob 234 (20B7) VHCH1-heavy nucleotidesequence, see Table 27 chain knob 235 (20B7) VHCH1-heavy see Table 27chain knob 236 (CLC-563) VHCH1-heavy nucleotide sequence, see Table 27chain knob 237 (CLC-563) VHCH1-heavy see Table 27 chain knob 238(CLC-564) VHCH1-heavy nucleotide sequence, see Table 27 chain knob 239(CLC-564) VHCH1-heavy see Table 27 chain knob 240 cynomolgus 4-1BB ECDaa 24-186 241 murine 4-1BB ECD P20334, aa 24-187 242 human 4-1BB antigenFc nucleotide sequence, see Table 37 knob chain 243 cynomolgus 4-1BBantigen nucleotide sequence, see Table 37 Fc knob chain 244 murine 4-1BBantigen Fc nucleotide sequence, see Table 37 knob chain 245 human 4-1BBantigen Fc see Table 37 knob chain 246 cynomolgus 4-1BB antigen seeTable 37 Fc knob chain 247 murine 4-1BB antigen Fc see Table 37 knobchain 248 Primer MS63 see Table 43 249 Nucleotide sequence see Table 444-1BB(12B3) VL 250 Nucleotide sequence see Table 44 4-1BB(12B3) VH 251Nucleotide sequence see Table 44 4-1BB(25G7) VL 252 Nucleotide sequencesee Table 44 4-1BB(25G7) VH 253 Nucleotide sequence see Table 444-1BB(11D5) VL 254 Nucleotide sequence see Table 44 4-1BB(11D5) VH 255Nucleotide sequence see Table 44 4-1BB(9B11) VL 256 Nucleotide sequencesee Table 44 4-1BB(9B11) VH 257 Nucleotide sequence see Table 444-1BB(20G2) VL 258 Nucleotide sequence see Table 44 4-1BB(20G2) VH 25912B3 P329GLALA IgG1 nucleotide sequence, see Table 45 (light chain) 26012B3 P329GLALA IgG1 nucleotide sequence, see Table 45 (heavy chain) 26112B3 P329GLALA IgG1 see Table 45 (light chain) 262 12B3 P329GLALA IgG1see Table 45 (heavy chain) 263 25G7 P329GLALA IgG1 nucleotide sequence,see Table 45 (light chain) 264 25G7 P329GLALA IgG1 nucleotide sequence,see Table 45 (heavy chain) 265 25G7 P329GLALA IgG1 see Table 45 (lightchain) 266 25G7 P329GLALA IgG1 see Table 45 (heavy chain) 267 11D5P329GLALA IgG1 nucleotide sequence, see Table 45 (light chain) 268 11D5P329GLALA IgG1 nucleotide sequence, see Table 45 (heavy chain) 269 11D5P329GLALA IgG1 see Table 45 (light chain) 270 11D5 P329GLALA IgG1 seeTable 45 (heavy chain) 271 9B11 P329GLALA IgG1 nucleotide sequence, seeTable 45 (light chain) 272 9B11 P329GLALA IgG1 nucleotide sequence, seeTable 45 (heavy chain) 273 9B11 P329GLALA IgG1 see Table 45 (lightchain) 274 9B11 P329GLALA IgG1 see Table 45 (heavy chain) 275 20G2P329GLALA IgG1 nucleotide sequence, see Table 45 (light chain) 276 20G2P329GLALA IgG1 nucleotide sequence, see Table 45 (heavy chain) 277 20G2P329GLALA IgG1 see Table 45 (light chain) 278 20G2 P329GLALA IgG1 seeTable 45 (heavy chain) 279 mu4-1BB D1/hu4-1BB see Table 56 D2 Fc knob280 hu4-1BB D1/mu4-1BB see Table 56 D2 Fc knob 281 hu4-1BB D1 Fc knobsee Table 56 282 Murine 4-1BB domain D1 see Table 57 (N-terminus) 283Human 4-1BB domain D2 see Table 57 (C-terminus) 284 Human 4-1BB domainD1 see Table 57 (N-terminus) 285 Murine 4-1BB domain D2 see Table 52(C-terminus) 286 (12B3) VHCH1-Heavy nucleotide sequence, see Table 60chain-(28H1) VHCL 287 (12B3) VHCH1-Heavy see Table 60 chain-(28H1) VHCL288 (25G7) VHCH1-Heavy nucleotide sequence, see Table 60 chain-(28H1)VHCL 289 (25G7) VHCH1-Heavy see Table 60 chain-(28H1) VHCL 290 (11D5)VHCH1-Heavy nucleotide sequence, see Table 60 chain-(28H1) VHCL 291(11D5) VHCH1-Heavy see Table 60 chain-(28H1) VHCL 292 (9B11) VHCH1-Heavynucleotide sequence, see Table 60 chain-(28H1) VHCL 293 (9B11)VHCH1-Heavy see Table 60 chain-(28H1) VHCL 294 (12B3) VHCH1-heavynucleotide sequence, see Table 65 chain knob 295 (12B3) VHCH1-heavy seeTable 65 chain knob 296 (25G7) VHCH1-heavy nucleotide sequence, seeTable 65 chain knob 297 (25G7) VHCH1-heavy see Table 65 chain knob 298(11D5) VHCH1-heavy nucleotide sequence, see Table 65 chain knob 299(11D5) VHCH1-heavy see Table 65 chain knob 300 (9B11) VHCH1-heavynucleotide sequence, see Table 65 chain knob 301 (9B11) VHCH1-heavy seeTable 65 chain knob 302 (8H9) VHCH1-heavy nucleotide sequence, see Table27 chain knob 303 (8H9) VHCH1-heavy see Table 27 chain knob 304 (49B4)VHCH1 Fc knob see Table 30 VH (4B9) (nucleotide sequence of heavychain 1) 305 (49B4) VHCH1 Fc hole see Table 30 VL (4B9) (nucleotidesequence of heavy chain 2) 306 (49B4) VHCH1 Fc knob see Table 30 VH(4B9) (heavy chain 1) 307 (49B4) VHCH1 Fc hole see Table 30 VL (4B9)(heavy chain 2) 308 (49B4) VHCH1 Fc knob see Table 30 VH (28H1)(nucleotide sequence, heavy chain 1) 309 (49B4) VHCH1 Fc hole see Table30 VL (28H1) (nucleotide sequence, heavy chain 2) 310 (49B4) VHCH1 Fcknob see Table 30 VH (28H1) (heavy chain 1) 311 (49B4) VHCH1 Fc hole seeTable 30 VL (28H1) (heavy chain 2) 312 (49B4) VHCH1 Fc knob see Table 30VH (DP47) (nucleotide sequence, heavy chain 1) 313 (49B4) VHCH1 Fc holesee Table 30 VL (DP47) (nucleotide sequence, heavy chain 2) 314 (49B4)VHCH1 Fc knob see Table 30 VH (DP47) (heavy chain 1) 315 (49B4) VHCH1 Fchole see Table 30 VL (DP47) (heavy chain 2) 316 (12B3) VHCH1 Fc knob seeTable 70 VH (4B9) (nucleotide sequence of HC 1) 317 (12B3) VHCH1 Fc holesee Table 70 VL (4B9) (nucleotide sequence of HC2) 318 (12B3) VHCH1 Fcknob see Table 70 VH (4B9) (heavy chain 1) 319 (12B3) VHCH1 Fc hole seeTable 70 VL (4B9) (heavy chain 2) 320 (25G7) VHCH1 Fc knob see Table 70VH (4B9) (nucleotide sequence, heavy chain 1) 321 (25G7) VHCH1 Fc holesee Table 70 VL (4B9) (nucleotide sequence, heavy chain 2) 322 (25G7)VHCH1 Fc knob see Table 70 VH (4B9) (heavy chain 1) 323 (25G7) VHCH1 Fchole see Table 70 VL (4B9) (heavy chain 2) 324 (11D5) VHCH1 Fc knob seeTable 70 VH (4B9) (nucleotide sequence, heavy chain 1) 325 (11D5) VHCH1Fc hole see Table 70 VL (4B9) (nucleotide sequence, heavy chain 2) 326(11D5) VHCH1 Fc knob see Table 70 VH (4B9) (heavy chain 1) 327 (11D5)VHCH1 Fc hole see Table 70 VL (4B9) (heavy chain 2) 328 (9B11) VHCH1 Fcknob see Table 70 VH (4B9) (nucleotide sequence, heavy chain 1) 329(9B11) VHCH1 Fc hole see Table 70 VL (4B9) (nucleotide sequence, heavychain 2) 330 (9B11) VHCH1 Fc knob see Table 70 VH (4B9) (heavy chain 1)331 (9B11) VHCH1 Fc hole see Table 70 VL (4B9) (heavy chain 2) 332(12B3) VHCH1 Fc knob see Table 71 VL (4B9) (nucleotide sequence of HC 1)333 (12B3) VHCH1 Fc hole see Table 71 VH (4B9) (nucleotide sequence ofHC2) 334 (12B3) VHCH1 Fc knob see Table 71 VL (4B9) (heavy chain 1) 335(12B3) VHCH1 Fc hole see Table 71 VH (4B9) (heavy chain 2) 336 (25G7)VHCH1 Fc knob see Table 71 VL (4B9) (nucleotide sequence, heavy chain 1)337 (25G7) VHCH1 Fc hole see Table 71 VH (4B9) (nucleotide sequence,heavy chain 2) 338 (25G7) VHCH1 Fc knob see Table 71 VL (4B9) (heavychain 1) 339 (25G7) VHCH1 Fc hole see Table 71 VH (4B9) (heavy chain 2)340 (11D5) VHCH1 Fc knob see Table 71 VL (4B9) (nucleotide sequence,heavy chain 1) 341 (11D5) VHCH1 Fc hole see Table 71 VH (4B9)(nucleotide sequence, heavy chain 2) 342 (11D5) VHCH1 Fc knob see Table71 VL (4B9) (heavy chain 1) 343 (11D5) VHCH1 Fc hole see Table 71 VH(4B9) (heavy chain 2) 344 (9B11) VHCH1 Fc knob see Table 71 VL (4B9)(nucleotide sequence, heavy chain 1) 345 (9B11) VHCH1 Fc hole see Table71 VH (4B9) (nucleotide sequence, heavy chain 2) 346 (9B11) VHCH1 Fcknob see Table 71 VL (4B9) (heavy chain 1) 347 (9B11) VHCH1 Fc hole seeTable 71 VH (4B9) (heavy chain 2)All nucleotide sequences are presented without the respective stop codonsequences.

In the following specific embodiments of the invention are listed:

1. A bispecific antigen binding molecule, comprising

-   -   (a) at least one moiety capable of specific binding to a        costimulatory TNF receptor family member,    -   (b) at least one moiety capable of specific binding to a target        cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

2. The bispecific antigen binding molecule of claim 1, wherein thecostimulatory TNF receptor family member is selected from the groupconsisting of OX40 and 4-1BB.

3. The bispecific antigen binding molecule of claims 1 or 2, wherein thecostimulatory TNF receptor family member is OX40.

4. The bispecific antigen binding molecule of any one of claims 1 to 3,wherein the moiety capable of specific binding to a costimulatory TNFreceptor family member binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.

5. The bispecific antigen binding molecule of any one of claims 1 to 4,comprising at least one moiety capable of specific binding to OX40,wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:2 and SEQ ID NO:3,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:4 and SEQ ID NO:5, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,        SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID        NO:15,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID        NO:18, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID        NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

6. The bispecific antigen binding molecule of any one of claims 1 to 5,wherein the moiety capable of specific binding to OX40 comprises a heavychain variable region VH comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and SEQ IDNO:37 and a light chain variable region VL comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence of SEQ ID NO:26, SEQ ID NO: 28, SEQID NO:30, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and SEQID NO:38.

7. The bispecific antigen binding molecule of any one of claims 1 to 5,wherein the moiety capable of specific binding to OX40 comprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

8. The bispecific antigen binding molecule of claims 1 or 2, wherein thecostimulatory TNF receptor family member is 4-1BB.

9. The bispecific antigen binding molecule of any one of claims 1, 2 or8, wherein the moiety capable of specific binding to a costimulatory TNFreceptor family member binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:39.

10. The bispecific antigen binding molecule of any one of claims 1, 2, 8or 9, comprising at least one moiety capable of specific binding to4-1BB, wherein said moiety comprises a VH domain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:40 and SEQ ID NO:41,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:42 and SEQ ID NO:43, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:44, SEQ ID NO:45, SEQ ID        NO:46, SEQ ID NO:47 and SEQ ID NO:48        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:49 and SEQ ID NO:50,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:51 and SEQ ID NO:52, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ ID        NO:55, SEQ ID NO:56 and SEQ ID NO:57.

11. The bispecific antigen binding molecule of any one of claims 1, 2,8, 9 or 10, wherein the moiety capable of specific binding to 4-1BBcomprises a heavy chain variable region VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 and SEQ IDNO:66 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 and SEQ IDNO:67.

12. The bispecific antigen binding molecule of any one of claims 1, 2and 8 to 11, wherein the moiety capable of specific binding to 4-1BBcomprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

13. The bispecific antigen binding molecule of any one of claims 1 to12, wherein the target cell antigen is selected from the groupconsisting of Fibroblast Activation Protein (FAP), Melanoma-associatedChondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth FactorReceptor (EGFR), Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.

14. The bispecific antigen binding molecule of any one of claims 1 to13, wherein the target cell antigen is Fibroblast Activation Protein(FAP).

15. The bispecific antigen binding molecule of any one of claims 1 to13, wherein the moiety capable of specific binding to FAP comprises a VHdomain comprising

-   -   (i) a CDR-H1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:68 and SEQ ID NO:69,    -   (ii) a CDR-H2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:70 and SEQ ID NO:71, and    -   (iii) a CDR-H3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:72 and SEQ ID NO:73,        and a VL domain comprising    -   (iv) a CDR-L1 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:74 and SEQ ID NO:75,    -   (v) a CDR-L2 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:76 and SEQ ID NO:77, and    -   (vi) a CDR-L3 comprising the amino acid sequence selected from        the group consisting of SEQ ID NO:78 and SEQ ID NO:79.

16. The bispecific antigen binding molecule of any one of claims 1 to 7,wherein

-   (i) the moiety capable of specific binding to OX40 comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29,    SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID    NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

17. The bispecific antigen binding molecule of any one of claims 1, 2 or8 to 12, wherein

-   (i) the moiety capable of specific binding to 4-1BB comprises a    heavy chain variable region VH comprising an amino acid sequence    that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to    the amino acid sequence of SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,    SEQ ID NO:64 or SEQ ID NO:66 and a light chain variable region    comprising an amino acid sequence that is at least about 95%, 96%,    97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID    NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67 and-   (ii) the moiety capable of specific binding to FAP comprises a heavy    chain variable region VH comprising an amino acid sequence that is    at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the    amino acid sequence of SEQ ID NO:80 or SEQ ID NO:82 and a light    chain variable region comprising an amino acid sequence that is at    least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino    acid sequence of SEQ ID NO:81 or SEQ ID NO:83.

18. The bispecific antigen binding molecule of any one of claims 1 to17, wherein said molecule comprises

-   -   (a) a first Fab fragment capable of specific binding to a        costimulatory TNF receptor family member,    -   (b) a second Fab fragment capable of specific binding to a        target cell antigen, and    -   (c) a Fc domain composed of a first and a second subunit capable        of stable association.

19. The bispecific antigen binding molecule of any one of claims 1 to18, wherein the Fc domain is an IgG, particularly an IgG1 Fc domain oran IgG4 Fc domain.

20. The bispecific antigen binding molecule of any one of claims 1 to19, wherein the Fc domain comprises one or more amino acid substitutionthat reduces the binding affinity of the antibody to an Fc receptorand/or effector function.

21. The bispecific antigen binding molecule of any one of claims 1 to20, wherein the Fc domain is of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex).

22. The bispecific antigen binding molecule of any one of claims 1 to21, wherein the Fc domain comprises a modification promoting theassociation of the first and second subunit of the Fc domain.

23. The bispecific antigen binding molecule of any one of claims 1 to22, wherein the first subunit of the Fc domain comprises knobs and thesecond subunit of the Fc domain comprises holes according to the knobsinto holes method.

24. The bispecific antibody of any one of claims 1 to 23, wherein thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (numbering according to Kabat EU index) and the secondsubunit of the Fc domain comprises the amino acid substitutions Y349C,T366S and Y407V (numbering according to Kabat EU index).

25. The bispecific antigen binding molecule of any one of claims 1 to24, comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) two moieties capable of specific binding to a target cell    antigen, and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

26. The bispecific antigen binding molecule of claim 25, wherein thebispecific antigen binding molecule is bivalent both for thecostimulatory TNF receptor family member and for the target cellantigen.

27. The bispecific antigen binding molecule of any one of claims 1 to24, comprising

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) two additional Fab fragments capable of specific binding to a    target cell antigen, wherein said additional Fab fragments are each    connected via a peptide linker to the C-terminus of the heavy chains    of (a).

28. The bispecific antigen binding molecule of claim 27, wherein the twoadditional Fab fragments capable of specific binding to a target cellantigen are crossover Fab fragments wherein the variable domains VL andVH are replaced by each other and the VL-CH chains are each connectedvia a peptide linker to the C-terminus of the heavy chains of (a).

29. The bispecific antigen binding molecule of claim 27 or 28, whereinthe two Fab fragments capable of specific binding to a costimulatory TNFreceptor family member are two Fab fragments capable of specific bindingto OX40 or 4-1BB and the two additional Fab fragments capable ofspecific binding to a target cell antigen are crossover Fab fragmentscapable of specific binding to FAP.

30. The bispecific antigen binding molecule of any one of the precedingclaims, comprising

-   (a) two moieties capable of specific binding to a costimulatory TNF    receptor family member,-   (b) one moiety capable of specific binding to a target cell antigen,    and-   (c) a Fc domain composed of a first and a second subunit capable of    stable association.

31. The bispecific antigen binding molecule of claim 30, wherein thebispecific antigen binding molecule is bivalent for the costimulatoryTNF receptor family member and monovalent for the target cell antigen.

32. The bispecific antigen binding molecule of any one of precedingclaims, comprising

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments capable of specific binding to a costimulatory TNF    receptor family member and the Fc domain, and-   (b) a VH and VL domain capable of specific binding to a target cell    antigen, wherein the VH domain is connected via a peptide linker to    the C-terminus of one of the heavy chains and wherein the VL domain    is connected via a peptide linker to the C-terminus of the second    heavy chain.

33. A polynucleotide encoding the bispecific antigen binding molecule ofany one of claims 1 to 32.

34. An antibody that specifically binds to OX40, wherein said antibodycomprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:25 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:26,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:27 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:28,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:29 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:30,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:31 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:32,    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:33 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:34,    -   (vi) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:35 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:36, or    -   (vii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:37 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:38.

35. An antibody that specifically binds to 4-1BB, wherein said antibodycomprises

-   -   (i) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:58 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:59,    -   (ii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:60 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:61,    -   (iii) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:62 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:63,    -   (iv) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:64 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:65, or    -   (v) a heavy chain variable region VH comprising an amino acid        sequence of SEQ ID NO:66 and and a light chain variable region        VL comprising an amino acid sequence of SEQ ID NO:67.

36. A polynucleotide encoding the antibody of claims 34 or 35.

37. A pharmaceutical composition comprising a bispecific antigen bindingmolecule of any one of claims 1 to 32 or an antibody of claims 34 or 35and at least one pharmaceutically acceptable excipient.

38. The bispecific antigen binding molecule of any one of claims 1 to32, or the antibody of claims 34 or 35, or the pharmaceuticalcomposition of claim 37, for use as a medicament.

39. The bispecific antigen binding molecule of any one of claims 1 to32, or the antibody of claim 34 or 35, or the pharmaceutical compositionof claim 37, for use

-   (i) in stimulating T cell response,-   (ii) in supporting survival of activated T cells,-   (iii) in the treatment of infections,-   (iv) in the treatment of cancer,-   (v) in delaying progression of cancer, or-   (vi) in prolonging the survival of a patient suffering from cancer.

40. The bispecific antigen binding molecule of any one of claims 1 to32, or the antibody of claims 34 or 35, or the pharmaceuticalcomposition of claim 37, for use in the treatment of cancer.

41. The bispecific antigen binding molecule of any one of claims 1 to32, or the antibody of claims 34 or 35, or the pharmaceuticalcomposition of claim 37, for use in the treatment of cancer, wherein thebispecific antigen binding molecule is administered in combination witha chemotherapeutic agent, radiation and/or other agents for use incancer immunotherapy.

42. A method of inhibiting the growth of tumor cells in an individualcomprising administering to the individual an effective amount of thebispecific antigen binding molecule of any one of claims 1 to 32, or theantibody of claims 34 or 35, or the pharmaceutical composition of claim37, to inhibit the growth of the tumor cells.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriatetemplates or were synthesized by Geneart AG (Regensburg, Germany) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.In cases where no exact gene sequence was available, oligonucleotideprimers were designed based on sequences from closest homologues and thegenes were isolated by RT-PCR from RNA originating from the appropriatetissue. The gene segments flanked by singular restriction endonucleasecleavage sites were cloned into standard cloning/sequencing vectors. Theplasmid DNA was purified from transformed bacteria and concentrationdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow sub-cloning into the respectiveexpression vectors. All constructs were designed with a 5′-end DNAsequence coding for a leader peptide which targets proteins forsecretion in eukaryotic cells.

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL or CH/CL exchange (CrossMabs) with emphasis ontheir correct assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/plasmin digested or alternativelydeglycosylated/limited LysC digested CrossMabs.

The CrossMabs were deglycosylated with N-Glycosidase F in a phosphate orTris buffer at 37° C. for up to 17 h at a protein concentration of 1mg/ml. The plasmin or limited LysC (Roche) digestions were performedwith 100 μg deglycosylated VH/VL CrossMabs in a Tris buffer pH 8 at roomtemperature for 120 hours and at 37° C. for 40 min, respectively. Priorto mass spectrometry the samples were desalted via HPLC on a SephadexG25 column (GE Healthcare). The total mass was determined via ESI-MS ona maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersaNanoMate source (Advion).

Example 1 Generation of Ox40 Antibodies and Tool Binders

1.1 Preparation, Purification and Characterization of Antigens andScreening Tools for the Generation of Novel OX40 Binders by PhageDisplay

DNA sequences encoding the ectodomains of human, mouse or cynomolgusOX40 (Table 1) were subcloned in frame with the human IgG1 heavy chainCH2 and CH3 domains on the knob (Merchant et al., 1998). An AcTEVprotease cleavage site was introduced between an antigen ectodomain andthe Fc of human IgG1. An Avi tag for directed biotinylation wasintroduced at the C-terminus of the antigen-Fc knob. Combination of theantigen-Fc knob chain containing the S354C/T366W mutations, with a Fchole chain containing the Y349C/T366S/L368A/Y407V mutations allowsgeneration of a heterodimer which includes a single copy of the OX40ectodomain containing chain, thus creating a monomeric form of Fc-linkedantigen (FIG. 1A). Table 1 shows the amino acid sequences of the variousOX40 ectodomains. Table 2 the cDNA and amino acid sequences of monomericantigen Fc(kih) fusion molecules as depicted in FIGS. 1A, 1B and 1C.

TABLE 1 Amino acid numbering of antigen ectodomains (ECD) and theirorigin SEQ ID NO: Construct Origin ECD 1 human OX40 ECD Synthetized aa29-214 according to P43489 122 cynomolgus OX40 ECD Isolated from aa29-214 cynomolgus blood 123 murine OX40 ECD Synthetized aa 10-211according to P47741

TABLE 2 cDNA and amino acid sequences of monomeric antigen Fc(kih)fusion molecules (produced by combination of one Fc hole chain with oneantigen Fc knob chain) SEQ ID NO: Antigen Sequence 124 NucleotideGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA sequenceCTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAC Fc hole chainCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAA 125 NucleotideCTGCACTGCGTGGGCGACACCTACCCCAGCAACGACCGG sequenceTGCTGCCACGAGTGCAGACCCGGCAACGGCATGGTGTCC human OX40CGGTGCAGCCGGTCCCAGAACACCGTGTGCAGACCTTGC antigen FcGGCCCTGGCTTCTACAACGACGTGGTGTCCAGCAAGCCCT knob chainGCAAGCCTTGTACCTGGTGCAACCTGCGGAGCGGCAGCGAGCGGAAGCAGCTGTGTACCGCCACCCAGGATACCGTGTGCCGGTGTAGAGCCGGCACCCAGCCCCTGGACAGCTACAAACCCGGCGTGGACTGCGCCCCTTGCCCTCCTGGCCACTTCAGCCCTGGCGACAACCAGGCCTGCAAGCCTTGGACCAACTGCACCCTGGCCGGCAAGCACACCCTGCAGCCCGCCAGCAATAGCAGCGACGCCATCTGCGAGGACCGGGATCCTCCTGCCACCCAGCCTCAGGAAACCCAGGGCCCTCCCGCCAGACCCATCACCGTGCAGCCTACAGAGGCCTGGCCCAGAACCAGCCAGGGGCCTAGCACCAGACCCGTGGAAGTGCCTGGCGGCAGAGCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACG AG 126 NucleotideCTCCACTGTGTCGGGGACACCTACCCCAGCAACGACCGGT sequenceGCTGTCAGGAGTGCAGGCCAGGCAACGGGATGGTGAGCC cynomolgusGCTGCAACCGCTCCCAGAACACGGTGTGCCGTCCGTGCG OX40 antigenGGCCCGGCTTCTACAACGACGTGGTCAGCGCCAAGCCCT Fc knob chainGCAAGGCCTGCACATGGTGCAACCTCAGAAGTGGGAGTGAGCGGAAACAGCCGTGCACGGCCACACAGGACACAGTCTGCCGCTGCCGGGCGGGCACCCAGCCCCTGGACAGCTACAAGCCTGGAGTTGACTGTGCCCCCTGCCCTCCAGGGCACTTCTCCCCGGGCGACAACCAGGCCTGCAAGCCCTGGACCAACTGCACCTTGGCCGGGAAGCACACCCTGCAGCCAGCCAGCAATAGCTCGGACGCCATCTGTGAGGACAGGGACCCCCCACCCACACAGCCCCAGGAGACCCAGGGCCCCCCGGCCAGGCCCACCACTGTCCAGCCCACTGAAGCCTGGCCCAGAACCTCACAGAGACCCTCCACCCGGCCCGTGGAGGTCCCCAGGGGCCCTGCGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 127 NucleotideGTGACCGCCAGACGGCTGAACTGCGTGAAGCACACCTAC sequenceCCCAGCGGCCACAAGTGCTGCAGAGAGTGCCAGCCCGGC murine OX40CACGGCATGGTGTCCAGATGCGACCACACACGGGACACC antigen FcCTGTGCCACCCTTGCGAGACAGGCTTCTACAACGAGGCCG knob chainTGAACTACGATACCTGCAAGCAGTGCACCCAGTGCAACCACAGAAGCGGCAGCGAGCTGAAGCAGAACTGCACCCCCA CCCAGGATACCGTGTGCAGATGCAGACCCGGCACCCAGCCCAGACAGGACAGCGGCTACAAGCTGGGCGTGGACTGCGTGCCCTGCCCTCCTGGCCACTTCAGCCCCGGCAACAACCAGGCCTGCAAGCCCTGGACCAACTGCACCCTGAGCGGCAAGCAGACCAGACACCCCGCCAGCGACAGCCTGGATGCCGTGTGCGAGGACAGAAGCCTGCTGGCCACCCTGCTGTGGGAGACACAGCGGCCCACCTTCAGACCCACCACCGTGCAGAGCACCACCGTGTGGCCCAGAACCAGCGAGCTGCCCAGTCCTCCTACCCTCGTGACACCTGAGGGCCCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAG ATTGAATGGCACGAG 128 Fc holechain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 129human OX40 LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGP antigen FcGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCR knob chainAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 130 cynomolgusLHCVGDTYPSNDRCCQECRPGNGMVSRCNRSQNTVCRPCG OX40 antigenPGFYNDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCR Fc knob chainCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPPARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 131 murine OX40VTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTL antigen FcCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQ knob chainDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE

All OX40-Fc-fusion encoding sequences were cloned into a plasmid vectordriving expression of the insert from an MPSV promoter and containing asynthetic polyA signal sequence located at the 3′ end of the CDS. Inaddition, the vector contained an EBV OriP sequence for episomalmaintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a2:1:0.05 ratio (“antigen ECD-AcTEV-Fc knob”:“Fc hole”:“BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were resuspended in 20 mLof CD CHO medium containing 200 μg of vector DNA. After addition of 540μL of polyethylenimine (PEI), the solution was vortexed for 15 secondsand incubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, 160 mL of F17 medium was added andcells were cultured for 24 hours. One day after transfection, 1 mMvalproic acid and 7% Feed were added to the culture. After 7 days ofculturing, the cell supernatant was collected by spinning down cells for15 min at 210 g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unboundprotein was removed by washing with at least 10 column volumes of abuffer containing 20 mM sodium phosphate, 20 mM sodium citrate and 0.5 Msodium chloride (pH 7.5). The bound protein was eluted using a linearpH-gradient of sodium chloride (from 0 to 500 mM) created over 20 columnvolumes of 20 mM sodium citrate, 0.01% (v/v) Tween-20, pH 3.0. Thecolumn was then washed with 10 column volumes of a solution containing20 mM sodium citrate, 500 mM sodium chloride and 0.01% (v/v) Tween-20,pH 3.0.

The pH of the collected fractions was adjusted by adding 1/40 (v/v) of2M Tris, pH8.0. The protein was concentrated and filtered prior toloading on a HiLoad Superdex 200 column (GE Healthcare) equilibratedwith 2 mM MOPS, 150 mM sodium chloride, 0.02% (w/v) sodium azidesolution of pH 7.4.

1.2 Selection of Ox40-specific 8119, 20B7, 49B4, 1G4, CLC-563, CLC-564and 17A9 Antibodies from Generic Fab and Common Light Chain Libraries

Anti-OX40 antibodies were selected from three different generic phagedisplay libraries: DP88-4 (clones 20B7, 8H9 1G4 and 49B4), the commonlight chain library Vk3_20/VH3_23 (clones CLC-563 and CLC-564) andlambda-DP47 (clone 17A9).

The DP88-4 library was constructed on the basis of human germline genesusing the V-domain pairing Vk1_5 (kappa light chain) and VH1_69 (heavychain) comprising randomized sequence space in CDR3 of the light chain(L3, 3 different lengths) and CDR3 of the heavy chain (H3, 3 differentlengths). Library generation was performed by assembly of 3PCR-amplified fragments applying splicing by overlapping extension (SOE)PCR. Fragment 1 comprises the 5′ end of the antibody gene includingrandomized L3, fragment 2 is a central constant fragment spanning fromL3 to H3 whereas fragment 3 comprises randomized H3 and the 3′ portionof the antibody gene. The following primer combinations were used togenerate these library fragments for DP88-4 library: fragment 1 (forwardprimer LMB3 combined with reverse primers Vk1_5_L3r_S or Vk1_5_L3r_SY orVk1_5_L3r_SPY), fragment 2 (forward primer RJH31 combined with reverseprimer RJH32) and fragment 3 (forward primers DP88-v4-4 or DP88-v4-6 orDP88-v4-8 combined with reverse primer fdseqlong), respectively. PCRparameters for production of library fragments were 5 min initialdenaturation at 94° C., 25 cycles of 1 min 94° C., 1 min 58° C., 1 min72° C. and terminal elongation for 10 min at 72° C. For assembly PCR,using equimolar ratios of the gel-purified single fragments as template,parameters were 3 min initial denaturation at 94° C. and 5 cycles of 30s 94° C., 1 min 58° C., 2 min 72° C. At this stage, outer primers (LMB3and fdseqlong) were added and additional 20 cycles were performed priorto a terminal elongation for 10 min at 72° C. After assembly ofsufficient amounts of full length randomized Fab constructs, they weredigested NcoI/NheI and ligated into similarly treated acceptor phagemidvector. Purified ligations were used for ˜60 transformations intoelectrocompetent E. coli TG1. Phagemid particles displaying the Fablibrary were rescued and purified by PEG/NaCl purification to be usedfor selections. These library construction steps were repeated threetimes to obtain a final library size of 4.4×109. Percentages offunctional clones, as determined by C-terminal tag detection in dotblot, were 92.6% for the light chain and 93.7% for the heavy chain,respectively.

The common light chain library Vk3_20/VH3_23 was constructed on thebasis of human germline genes using the V-domain pairing Vk3_20 (kappalight chain) and VH3_23 (heavy chain) comprising a constantnon-randomized common light chain Vk3_20 and randomized sequence spacein CDR3 of the heavy chain (H3, 3 different lengths). Library generationwas performed by assembly of 2 PCR-amplified fragments applying splicingby overlapping extension (SOE) PCR. Fragment 1 is a constant fragmentspanning from L3 to H3 whereas fragment 2 comprises randomized H3 andthe 3′ portion of the antibody gene. The following primer combinationswere used to generate these library fragments for the Vk3_20/VH3_23common light chain library: fragment 1 (forward primer MS64 combinedwith reverse primer DP47CDR3_ba (mod.)) and fragment 2 (forward primersDP47-v4-4, DP47-v4-6, DP47-v4-8 combined with reverse primer fdseqlong),respectively. PCR parameters for production of library fragments were 5min initial denaturation at 94° C., 25 cycles of 1 min 94° C., 1 min 58°C., 1 min 72° C. and terminal elongation for 10 min at 72° C. Forassembly PCR, using equimolar ratios of the gel-purified singlefragments as template, parameters were 3 min initial denaturation at 94°C. and 5 cycles of 30 s 94° C., 1 min 58° C., 2 min 72° C. At thisstage, outer primers (MS64 and fdseqlong) were added and additional 18cycles were performed prior to a terminal elongation for 10 min at 72°C. After assembly of sufficient amounts of full length randomized VHconstructs, they were digested MunI/NotI and ligated into similarlytreated acceptor phagemid vector. Purified ligations were used for ˜60transformations into electrocompetent E. coli TG1. Phagemid particlesdisplaying the Fab library were rescued and purified by PEG/NaClpurification to be used for selections. A final library size of 3.75×109was obtained. Percentages of functional clones, as determined byC-terminal tag detection in dot blot, were 98.9% for the light chain and89.5% for the heavy chain, respectively.

The lambda-DP47 library was constructed on the basis of human germlinegenes using the following V-domain pairings: Vl3_19 lambda light chainwith VH3_23 heavy chain. The library was randomized in CDR3 of the lightchain (L3) and CDR3 of the heavy chain (H3) and was assembled from 3fragments by “splicing by overlapping extension” (SOE) PCR. Fragment 1comprises the 5′ end of the antibody gene including randomized L3,fragment 2 is a central constant fragment spanning from the end of L3 tothe beginning of H3 whereas fragment 3 comprises randomized H3 and the3′ portion of the Fab fragment. The following primer combinations wereused to generate library fragments for library: fragment 1(LMB3-Vl_3_19_L3r_V/Vl_3_19_L3r_HV/Vl_3_19_L3r_HLV), fragment 2(RJH80-DP47CDR3_ba (mod)) and fragment 3(DP47-v4-4/DP47-v4-6/DP47-v4-8-fdseqlong). PCR parameters for productionof library fragments were 5 min initial denaturation at 94° C., 25cycles of 60 sec at 94° C., 60 sec at 55° C., 60 sec at 72° C. andterminal elongation for 10 min at 72° C. For assembly PCR, usingequimolar ratios of the 3 fragments as template, parameters were 3 mininitial denaturation at 94° C. and 5 cycles of 60 sec at 94° C., 60 secat 55° C., 120 sec at 72° C. At this stage, outer primers were added andadditional 20 cycles were performed prior to a terminal elongation for10 min at 72° C. After assembly of sufficient amounts of full lengthrandomized Fab fragments, they were digested with NcoI/NheI alongsidewith similarly treated acceptor phagemid vector. 15 ug of Fab libraryinsert were ligated with 13.3 ug of phagemid vector. Purified ligationswere used for 60 transformations resulting in 1.5×10⁹ transformants.Phagemid particles displaying the Fab library were rescued and purifiedby PEG/NaCl purification to be used for selections.

Human OX40 (CD134) as antigen for the phage display selections wastransiently expressed as N-terminal monomeric Fc-fusion in HEK EBNAcells and in vivo site-specifically biotinylated via co-expression ofBirA biotin ligase at the avi-tag recognition sequence located a theC-terminus of the Fc portion carrying the receptor chain (Fc knobchain).

Selection rounds (biopanning) were performed in solution according tothe following pattern:

-   1. Pre-clearing of ˜1012 phagemid particles on maxisorp plates    coated with 10 ug/ml of an unrelated human IgG to deplete the    libraries of antibodies recognizing the Fc-portion of the antigen,-   2. incubation of the non-binding phagemid particles with 100 nM    biotinylated human OX40 for 0.5 h in the presence of 100 nM    unrelated non-biotinylated Fc knob-into-hole construct for further    depletion of Fc-binders in a total volume of 1 ml,-   3. capture of biotinylated hu OX40 and attached specifically binding    phage by transfer to 4 wells of a neutravidin pre-coated microtiter    plate for 10 min (in rounds 1 & 3),-   4. washing of respective wells using 5×PBS/Tween20 and 5×PBS,-   5. elution of phage particles by addition of 250 ul 100 mM TEA    (triethylamine) per well for 10 min and neutralization by addition    of 500 ul 1M Tris/HCl pH 7.4 to the pooled eluates from 4 wells,-   6. post-clearing of neutralized eluates by incubation on neutravidin    pre-coated microtiter plate with 100 nM biotin-captured Fc    knob-into-hole construct for final removal of Fc-binders,-   7. re-infection of log-phase E. coli TG1 cells with the supernatant    of eluted phage particles, infection with helperphage VCSM13,    incubation on a shaker at 30° C. over night and subsequent PEG/NaCl    precipitation of phagemid particles to be used in the next selection    round.

Selections were carried out over 3 or 4 rounds using constant antigenconcentrations of 100 nM. In order to increase the likelihood forbinders that are cross-reactive not only to cynomolgus OX40 but alsomurine OX40, in some selection rounds the murine target was used insteadof the human OX40. In rounds 2 and 4, in order to avoid enrichment ofbinders to neutravidin, capture of antigen:phage complexes was performedby addition of 5.4×107 streptavidin-coated magnetic beads. Specificbinders were identified by ELISA as follows: 100 ul of 25 nMbiotinylated human OX40 and 10 ug/ml of human IgG were coated onneutravidin plates and maxisorp plates, respectively. Fab-containingbacterial supernatants were added and binding Fabs were detected viatheir Flag-tags using an anti-Flag/HRP secondary antibody. Clonesexhibiting signals on human OX40 and being negative on human IgG wereshort-listed for further analyses and were also tested in a similarfashion against cynomolgus and murine OX40. They were bacteriallyexpressed in a 0.5 liter culture volume, affinity purified and furthercharacterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.

Table 3 shows the sequence of generic phage-displayed antibody library(DP88-4), Table 4 provides cDNA and amino acid sequences of libraryDP88-4 germline template and Table 5 shows the Primer sequences used forgeneration of DP88-4 germline template.

TABLE 3 Sequence of generic phage-displayed antibody library (DP88-4)SEQ ID NO: Description Sequence 132 nucleotideTGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCG sequence ofCGGCCCAGCCGGCCATGGCCGACATCCAGATGACCCAGTCTCCT pRJH33TCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGC libraryCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCA templateGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCA DP88-4GTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCC library;GGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGA completeTTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTACGTT Fab codingTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCA regionCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT comprisingGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA PelB leaderGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG sequence +GTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG Vk1_5CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT kappa V-ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG domain +CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTG CL constantGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGG domain forAGCCGCAGACTACAAGGACGACGACGACAAGGGTGCCGCATAA light chainTAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATA and PelB +CCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCA VH1_69 V-GCCGGCGATGGCCCAGGTGCAATTGGTGCAGTCTGGGGCTGAGG domain +TGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC CH1GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGC constantCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCT domain forTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACC heavy chainATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAG includingCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAC tagsTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCGCGGCA

TABLE 4 cDNA and amino acid sequences of library DP88-4 germlinetemplate SEQ ID NO: Description Sequence 133 nucleotideGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC sequence ofATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCA Fab light chainGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAG Vk1_5AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGG TGCCGCA 134 Fab light chainDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP Vk1_5GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGAAEQKLISEEDLNGAADYKDDDDK GAA 135 nucleotideCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA sequence ofAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC Fab heavy chainGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC VH1_69GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCG CGGCA 136 Fab heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VH1_69APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDAAASTSAHHHH HHAAA

TABLE 5 Primer sequences used for generation of DP88-4 library SEQ IDNO: Primer name Primer sequence 5′-3′ 137 LMB3CAGGAAACAGCTATGACCATGATTAC 138 Vk1_5_L3r_S CTCGACTTTGGTGCCCTGGCCAAACGTS

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCAT underlined: 60% original base and 40%randomization as M. bolded and italic: 60% original base and 40%randomization as N 139 Vk1_5_L3r_SY CTCGACTTTGGTGCCCTGGCCAAACGTM

S

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCAT underlined: 60% original base and 40%randomization as M. bolded and italic: 60% original base and 40%randomization as N 140 Vk1_5_L3r_SPY CTCGACTTTGGTGCCCTGGCCAAACGTM

M SS S

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCAT underlined: 60% original base and 40%randomization as M. bolded and italic: 60% original base and 40%randomization as N 141 RJH31 ACGTTTGGCCAGGGCACCAAAGTCGAG 142 RJH32TCTCGCACAGTAATACACGGCGGTGTCC 143 DP88-v4-4GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3:G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%. 144DP88-v4-6 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3:G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%. 145 DP88-v4-8 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3:G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%. 146fdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG

Table 6 shows the sequence of generic phage-displayed antibody commonlight chain library (Vk3_20/VH3_23). Table 7 provides cDNA and aminoacid sequences of common light chain library (Vk3_20/VH3_23) germlinetemplate and Table 8 shows the Primer sequences used for generation ofcommon light chain library (Vk3_20/VH3_23).

TABLE 6 Sequence of generic phage-displayed antibody common light chainlibrary (Vk3_20/VH3_23) template used for PCR SEQ ID NO: DescriptionSequence 147 pRJH110 ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTClibrary GCGGCCCAGCCGGCCATGGCCGAAATCGTGTTAACGCAGTCTCC template ofAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCTT commonGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTAC light chainCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGC libraryATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTG Vk3_20/VH3_23;GATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCT completeGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC Fab codingGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACG regionGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAG comprisingTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTC PelB leaderTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC sequence +TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG Vk3_20CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC kappa V-AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA domain +CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG CLGGAGAGTGTGGAGCCGCACATCACCATCACCATCACGGAGCCG constantCAGACTACAAGGACGACGACGACAAGGGTGCCGCATAATAAGG domain forCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATACCTGC light chainTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGG and PelB +CGATGGCCGAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTA VH3_23CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATT V-domain +CACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAG CH1GGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGT constantAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTC domain forCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCC heavyTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCGTTT chainCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTC includingGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCT tagsCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGA TCTGAATGCCGCGGCA

TABLE 7 cDNA and amino acid sequences of common light chain library(Vk3_20/VH3_23) germline template SEQ ID NO: Description Sequence 148nucleotide GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGT sequence ofCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTC Fab light chainAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGA Vk3_20AACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCACATCACCATCACCATCACGGAGCCGCAGACTACAAGGACGACGACGACAAGGGT GCCGCA 149 Fab light chainEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPG Vk3_20QAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECGAAHHHHHHGAADYKDDDDKGAA150 nucleotide GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAG sequence ofCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT Fab heavy chainTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC VH3_23TCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGCCGCGGCA 151 Fab heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP VH3_23 (DP47)GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDAAAEQKLISEEDLNAAA

TABLE 8 Primer sequences used for generation of DP88-4 library SEQ IDNO: Primer name Primer sequence 5′-3′ 152 MS64 ACGTTCGGCCAGGGGACCAAAGTGG153 DP47CDR3_ba CGCACAGTAATATACGGCCGTGTCC (mod.) 154 DP47-v4-4CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 155 DP47-v4-6CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 156 DP47-v4-8CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 157 fdseqlongGACGTTAGTAAATGAATTTTCTGTATGAGG 1: G/D = 20%, E/V/S = 10%, A/P/R/L/T/Y= 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3: G/A/Y = 20%,P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%; 5: K = 70%, R = 30%.

Table 9 shows the sequence of generic phage-displayed lambda-DP47library (Vl3_19/VH3_23) template used for PCRs. Table 10 provides cDNAand amino acid sequences of lambda-DP47 library (Vl3_19/VH3_23) germlinetemplate and Table 11 shows the Primer sequences used for generation oflambda-DP47 library (Vl3_19/VH3_23).

TABLE 9 Sequence of generic phage-displayed lambda-DP47 library(Vl3_19/VH3_23) template used for PCRs SEQ ID NO: Description Sequence158 pRJH53 ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTC libraryGCGGCCCAGCCGGCCATGGCCTCGTCTGAGCTGACTCAGGACCC template ofTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCC lambda-AAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAG DP47AAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAA libraryCCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAG Vl3_19/VH3_23;GAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT completeGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGCGGTAATCA Fab codingTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAAC regionCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAG comprisingGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGA PelB leaderCTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCA sequence +GCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCA Vl3_19GAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACC lambda V-CCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGA domain +CCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGA CL constantGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGAT domain forCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTG light chainCCGCATAATAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCAT and PelB +ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTC VH3_23 V-GCTGCCCAGCCGGCGATGGCCGAGGTGCAATTGCTGGAGTCTGG domain +GGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CH1CAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCC constantGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGT domain forGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG heavy chainGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC includingAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT tagsGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCA TCACCATCACCATCACGCCGCGGCA

TABLE 10 cDNA and amino acid sequences of lambda-DP47 library(Vl3_19/VH3_23) germline template SEQ ID NO: Description Sequence 159nucleotide TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCT sequence ofTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCC Fab light chainTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAG Vl3_19GACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGCGGTAATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGAC GACGACAAGGGTGCCGCA 160 Fab lightchain SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQ Vl3_19APVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGAAEQKLISEEDLNGAADYKDDDDKGAA 150 nucleotide see Table 7 sequenceof Fab heavy chain VH3_23 151 Fab heavy chain see Table 7 VH3_23 (DP47)

TABLE 11 Primer sequences used for generation of lambda-DP47 library(Vl3_19/VH3_23) SEQ ID NO: Primer name Primer sequence 5′-3′ 161 LMB3CAGGAAACAGCTATGACCATGATTAC 162 Vl_3_19_L3r_VGGACGGTCAGCTTGGTCCCTCCGCCGAATAC V

 A

 A

G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGC underlined: 60% original base and 40%randomization as M bold and italic: 60% original base and 40%randomization as N 163 Vl_3_19_L3r_HV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC C

 A

A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGC underlined: 60% original base and 40%randomization as M bolded and italic: 60% original base and 40%randomization as N 164 Vl_3_19_L3r_HLV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC R

 V

A

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTC CGC underlined: 60% original base and 40%randomization as M bolded and italic: 60% original base and 40%randomization as N 165 RJH80 TTCGGCGGAGGGACCAAGCTGACCGTCCAdditional primers used for construction of the lambda-DP47 library,i.e. DP47CDR3_ba (mod.), DP47-v4-4, DP47-v4-6, DP47-v4-8 and fdseqlong,are identical to the primers used for the construction of the commonlight chain library (Vk3_20/VH3_23) and have already been listed inTable 8.

Clones 8H9, 20B7, 49B4, 1G4, CLC-563, CLC-564 and 17A9 were identifiedas human Ox40-specific binders through the procedure described above.The cDNA sequences of their variable regions are shown in Table 12below, the corresponding amino acid sequences can be found in Table C.

TABLE 12 Variable region base pair sequences for phage-derived anti-Ox40antibodies. Underlined are the complementarity determining regions(CDRs). SEQ ID Clone NO: Sequence 8H9 166 (VL)TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGTTATGCCTCATAATCGCGTATTCGGCGGAGGGACCAAGCTGACCGTC 167 (VH)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGTTTTCTACCGTGGTGGTGTTTCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 49B4 168 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAGTTCGCAGCCGTATACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 169 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 1G4 170 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATATTTCGTATTCCATGTTGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 171 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTCTATGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 20B7 172 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATCAGGCTTTTTCGCTTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 173 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGTTAACTACCCGTACTCTTACTGGGGTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA CLC- 174 (VL)GAGATCGTGCTGACCCAGAGCCCCGGCACACTCTCCCTGTCTCCTG 563GGGAAAGGGCCACCCTTTCATGCAGAGCCAGCCAGTCCGTCTCTAGTAGCTACCTGGCATGGTATCAGCAGAAGCCAGGACAAGCCCCCCGCCTCCTGATTTACGGCGCTTCCTCTCGGGCAACTGGTATCCCTGACAGGTTCTCAGGGAGCGGAAGCGGAACAGATTTTACCTTGACTATTTCTAGACTGGAGCCAGAGGACTTCGCCGTGTATTACTGTCAGCAGTACGGTAGTAGCCCCCTCACCTTTGGCCAGGGGACAAAAGTCGAAATCAAG 175 (VH)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCTTGACGTTGGTGCTTTCGACTACTGGGGCCAA GGAGCCCTGGTCACCGTCTCGAGTCLC- 176 (VL) GAGATCGTGCTGACCCAGAGCCCCGGCACACTCTCCCTGTCTCCTG 564GGGAAAGGGCCACCCTTTCATGCAGAGCCAGCCAGTCCGTCTCTAGTAGCTACCTGGCATGGTATCAGCAGAAGCCAGGACAAGCCCCCCGCCTCCTGATTTACGGCGCTTCCTCTCGGGCAACTGGTATCCCTGACAGGTTCTCAGGGAGCGGAAGCGGAACAGATTTTACCTTGACTATTTCTAGACTGGAGCCAGAGGACTTCGCCGTGTATTACTGTCAGCAGTACGGTAGTAGCCCCCTCACCTTTGGCCAGGGGACAAAAGTCGAAATCAAG 177 (VH)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGTTCGACGTTGGTCCGTTCGACTACTGGGGCCAA GGAACCCTGGTCACCGTCTCGAGT17A9 178 (VL) TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGTTATGCCTCATAATCGCGTATTCGGCGGAGGGACCAAGCTGACCGTC 179 (VH)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGTTTTCTACCGTGGTGGTGTTTCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT1.3 Preparation, Purification and Characterization of Anti-Ox40 IgG1P329G LALA Antibodies

The variable regions of heavy and light chain DNA sequences of selectedanti-Ox40 binders were subcloned in frame with either the constant heavychain or the constant light chain of human IgG1. The Pro329Gly,Leu234Ala and Leu235Ala mutations have been introduced in the constantregion of the knob and hole heavy chains to abrogate binding to Fc gammareceptors according to the method described in International PatentAppl. Publ. No. WO 2012/130831 A1.

The cDNA and amino acid sequences of the anti-Ox40 clones are shown inTable 13. All anti-Ox40-Fc-fusion encoding sequences were cloned into aplasmid vector, which drives expression of the insert from an MPSVpromoter and contains a synthetic polyA signal sequence located at the3′ end of the CDS. In addition, the vector contains an EBV OriP sequencefor episomal maintenance of the plasmid.

TABLE 13 Sequences of anti-Ox40 clones in P329GLALA human IgG1 formatClone SEQ ID No. Sequence 8B9 180GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATTTGACGTATTCGCGGTTTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 181CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTGGATGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAA 182DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYLTYSRFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 183QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49B4 184GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAGTTCGCAGCCGTATACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 185CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA 186DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSQPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 187QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1G4 188GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATATTTCGTATTCCATGTTGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 189CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTCTATGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAA 190DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYISYSMLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 191QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGSMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20B7 192GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATCAGGCTTTTTCGCTTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 193CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGTTAACTACCCGTACTCTTACTGGGGTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAAA194 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQAFSLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 195QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARVNYPYSYWGDFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK CLC- 196GAGATCGTGCTGACCCAGAGCCCCGGCACACTCTCCCTGTCTC 563 (nucleotideCTGGGGAAAGGGCCACCCTTTCATGCAGAGCCAGCCAGTCCGT sequence lightCTCTAGTAGCTACCTGGCATGGTATCAGCAGAAGCCAGGACAA chain)GCCCCCCGCCTCCTGATTTACGGCGCTTCCTCTCGGGCAACTGGTATCCCTGACAGGTTCTCAGGGAGCGGAAGCGGAACAGATTTTACCTTGACTATTTCTAGACTGGAGCCAGAGGACTTCGCCGTGTATTACTGTCAGCAGTACGGTAGTAGCCCCCTCACCTTTGGCCAGGGGACAAAAGTCGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT 197GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG (nucleotideGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTT sequence heavyAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG chain)GGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCTTGACGTTGGTGCTTTCGACTACTGGGGCCAAGGAGCCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCGGGTAAA 198EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR (Light chain)LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 199EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG (Heavy chain)LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CLC- 200GAGATCGTGCTGACCCAGAGCCCCGGCACACTCTCCCTGTCTC 564 (nucleotideCTGGGGAAAGGGCCACCCTTTCATGCAGAGCCAGCCAGTCCGT sequence lightCTCTAGTAGCTACCTGGCATGGTATCAGCAGAAGCCAGGACAA chain)GCCCCCCGCCTCCTGATTTACGGCGCTTCCTCTCGGGCAACTGGTATCCCTGACAGGTTCTCAGGGAGCGGAAGCGGAACAGATTTTACCTTGACTATTTCTAGACTGGAGCCAGAGGACTTCGCCGTGTATTACTGTCAGCAGTACGGTAGTAGCCCCCTCACCTTTGGCCAGGGGACAAAAGTCGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT 201GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG (nucleotideGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTT sequence heavyAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG chain)GGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGTTCGACGTTGGTCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCGGGTAAA 202EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR (Light chain)LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 203EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG (Heavy chain)LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAFDVGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 17A9 204TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGG (nucleotideACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAG sequence lightTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCT chain)GTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGTTATGCCTCATAATCGCGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 205GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG (nucleotideGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTT sequence heavyAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG chain)GGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGTTTTCTACCGTGGTGGTGTTTCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 206SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPV (Light chain)LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRVMPHNRVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 207EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG (Heavy chain)LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVFYRGGVSMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

The anti-Ox40 antibodies were produced by co-transfecting HEK293-EBNAcells with the mammalian expression vectors using polyethylenimine. Thecells were transfected with the corresponding expression vectors in a1:1 ratio (“vector heavy chain”: “vector light chain”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes at 210×g, and the supernatant was replaced bypre-warmed CD CHO medium. Expression vectors (200 μg of total DNA) weremixed in 20 mL CD CHO medium. After addition of 540 μL PEI, the solutionwas vortexed for 15 seconds and incubated for 10 minutes at roomtemperature. Afterwards, cells were mixed with the DNA/PEI solution,transferred to a 500 mL shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. After the incubation, 160 mLof F17 medium was added and cells were cultured for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed with supplements wereadded. After culturing for 7 days, the supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of antibody molecules from cell culture supernatants wascarried out by affinity chromatography using Protein A as describedabove for purification of antigen Fc fusions.

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl solution of pH 6.0.

The protein concentration of purified antibodies was determined bymeasuring the OD at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the antibodies were analyzed by CE-SDS in the presence andabsence of a reducing agent (Invitrogen, USA) using a LabChipGXII(Caliper). The aggregate content of antibody samples was analyzed usinga TSKgel G3000 SW XL analytical size-exclusion column (Tosoh)equilibrated in a 25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

Table 14 summarizes the yield and final content of the anti-Ox40 P329GLALA IgG1 antibodies.

TABLE 14 Biochemical analysis of anti-Ox40 P329G LALA IgG1 clones YieldMonomer Clone [mg/l] [%] CE-SDS (non red) CE-SDS (red) 8H9 P329GLALA 7100 1.2% (176 kDa) 66.9% (54 kDa) IgG1 96.1% (158 kDa) 28.9% (25 kDa)1.3% (142 kDa) 49B4 P329GLALA 7.5 100 99% (163 kDa) 81% (61.7 kDa) IgG11% (149 kDa) 18% (28.9 kDa) 1G4 P329GLALA 1 100 98.9% (167.4 kDa) 80%(63.4 kDa) IgG1 1.1% (151 kDa) 19% (28.9 kDa) 20B7 P329GLALA 17 93 97.9%(174 kDa) 79.8% (65.4 kDa) IgG1 19.9% (29.5 kDa) CLC-563 P329GLALA 6.2100 97.7% (160 kDa) 77.7% (60 kDa) IgG1 19.8% (26.4 kDa) CLC-564P329GLALA 13.5 100 98.4% (155 kDa) 79.3% (60.1 kDa) IgG1 19.8% (26.5kDa) 17A9 P329GLALA 7.5 100 98.6% (175 kDa) 74.1% (61 kDa) IgG1 1.4%(153 kDa) 25.5% (38 kDa)

Example 2 Characterization of Anti-OX40 Antibodies

2.1 Binding on Human OX40

2.1.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of phage-derived OX40-specific antibodies to the recombinantOX40 Fc(kih) was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 at 25° C. with HBS-EP asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%Surfactant P20, Biacore, Freiburg/Germany).

In the same experiment, the species selectivity and the avidity of theinteraction between the phage display derived anti-OX40 clones 8H9,49B4, 1G4, 20B7, CLC-563, CLC-564 and 17A9 (all human IgG1 P329GLALA),and recombinant OX40 (human, cyno and murine) was determined.Biotinylated human, cynomolgus and murine OX40 Fc(kih) were directlycoupled to different flow cells of a streptavidin (SA) sensor chip.Immobilization levels up to 600 resonance units (RU) were used.

Phage display derived anti-OX40 human IgG1 P329GLALA antibodies werepassed at a concentration range from 2 to 500 nM (3-fold dilution) witha flow of 30 μL/minute through the flow cells over 120 seconds. Complexdissociation was monitored for 210 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained in areference flow cell, where no protein was immobilized.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration and used to estimatequalitatively the avidity (Table 16).

In the same experiment, the affinities of the interaction between phagedisplay derived antibodies 8H9, 49B4, 1G4, 20B7, CLC-563 and CLC-564(human IgG1 P329GLALA) to recombinant OX40 were determined. For thispurpose, the ectodomain of human or murine Ox40 was also subcloned inframe with an avi (GLNDIFEAQKIEWHE) and a hexahistidine tag (for thesequences see Table 15) or obtained by cleavage with AcTEV protease andremoval of Fc by chromatographical method.

TABLE 15 Nucleotide and amino acid sequences of monomeric human andmurine OX40 His tag SEQ ID NO: Antigen Sequence 208 human OX40 Hisnucleotide sequence 209 human OX40 HisLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVDEQLYFQGGS GLNDIFEAQKIEWHEARAHHHHHH 210murine OX40 His nucleotide sequence 211 murine OX40 HisTARRLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRSLLATLLWETQRPTFRPTTVQSTTVWPRTSELPSPPTLVTPEGPVDEQLYFQGGSGLNDIFEAQKIEWHEARAHHHHHH

Protein production was performed as described above for the Fc-fusionprotein. Secreted proteins were purified from cell culture supernatantsby chelating chromatography, followed by size exclusion chromatography.

The first chromatographic step was performed on a NiNTA SuperflowCartridge (5 ml, Qiagen) equilibrated in 20 mM sodium phosphate, 500 nMsodium chloride, pH7.4. Elution was performed by applying a gradientover 12 column volume from 5% to 45% of elution buffer (20 mM sodiumphosphate, 500 nM sodium chloride, 500 mM Imidazole, pH7.4).

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 75 column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mMsodium chloride, 0.02% (w/v) sodium azide solution of pH 7.4.

Affinity determination was performed using two setups.

Setup 1) Anti-human Fab antibody (Biacore, Freiburg/Germany) wasdirectly coupled on a CM5 chip at pH 5.0 using the standard aminecoupling kit (Biacore, Freiburg/Germany). The immobilization level wasapproximately 9000 RU. Phage display derived antibodies to OX40 werecaptured for 60 seconds at concentrations of 25 to 50 nM. Recombinanthuman OX40 Fc(kih) was passed at a concentration range from 4 to 1000 nMwith a flow of 30 μL/minutes through the flow cells over 120 seconds.The dissociation was monitored for 120 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell. Here, the antigens were flown over a surface withimmobilized anti-human Fab antibody but on which HBS-EP has beeninjected rather than the antibodies.

Setup 2) Anti-human Fc antibody (Biacore, Freiburg/Germany) was directlycoupled on a CM5 chip at pH 5.0 using the standard amine coupling kit(Biacore, Freiburg/Germany). The immobilization level was approximately8000 RU. Phage display derived antibodies to Ox40 were captured for 60seconds at concentrations of 20 nM. Recombinant human Ox40 avi His waspassed at a concentration range from 2.3 to 600 nM with a flow of 30μL/minutes through the flow cells over 120 seconds. The dissociation wasmonitored for 120 seconds. Bulk refractive index differences werecorrected for by subtracting the response obtained on reference flowcell. Here, the antigens were flown over a surface with immobilizedanti-human Fab antibody but on which HBS-EP has been injected ratherthan the antibodies.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration.

Clones 49B4, 1G4 and CLC-564 bind human Ox40 Fc(kih) with a loweraffinity than clones 8H9, 20B7 and CLC-563.

Affinity constants for the interaction between anti-OX40 P329GLALA IgG1and human OX40 Fc(kih) were determined by fitting to a 1:1 Langmuirbinding.

TABLE 16 Binding of anti-OX40 antibodies to recombinant human OX40Recombinant human OX40 Recombinant human OX40 His Recombinant Fc(kih)(affinity format) (affinity format) human OX40 ka kd KD ka kd KD Clone(avidity format) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) 8H9 ++++ 1.6E+054.5E−03 2.8E−08 6.5E+04 2.0E−03 3.1E−08 49B4 ++ 2.5E+05 1.3E−01 5.1E−071.4E+06 6.7E−01 4.6E−07 1G4 ++ 3.0E+05 8.4E−08 2.8E−07 2.3E+06 5.7E−012.5E−07 20B7 +++ 3.2E+04 1.3E−03 4.2E−08 1.2E+05 6.6E−04 5.6E−09 CLC-563++ 3.6E+04 3.2E−03 8.9E−08 4.0E+04 3.6E−03 8.9E−08 CLC-564 ++++ 3.2E+044.2E−03 1.3E−07 3.8E+05 5.3E−03 1.4E−082.1.2 Binding to Human Ox40 Expressing Cells: Naïve and Activated HumanPeripheral Mononuclear Blood Leukocytes (PBMC)

Buffy coats were obtained from the Zurich blood donation center. Toisolate fresh peripheral blood mononuclear cells (PBMCs) the buffy coatwas diluted with the same volume of DPBS (Gibco by Life Technologies,Cat. No. 14190 326). 50 mL polypropylene centrifuge tubes (TPP, Cat.-No.91050) were supplied with 15 mL Histopaque 1077 (SIGMA Life Science,Cat.-No. 10771, polysucrose and sodium diatrizoate, adjusted to adensity of 1.077 g/mL) and the buffy coat solution was layered above theHistopaque 1077. The tubes were centrifuged for 30 min at 400×g, roomtemperature and with low acceleration and no break. Afterwards the PBMCswere collected from the interface, washed three times with DPBS andresuspended in T cell medium consisting of RPMI 1640 medium (Gibco byLife Technology, Cat. No. 42401-042) supplied with 10% Fetal BovineSerum (FBS, Gibco by Life Technology, Cat. No. 16000-044, Lot 941273,gamma-irradiated, mycoplasma-free and heat inactivated at 56° C. for 35min), 1% (v/v) GlutaMAX I (GIBCO by Life Technologies, Cat. No. 35050038), 1 mM Sodium-Pyruvat (SIGMA, Cat. No. S8636), 1% (v/v) MEMnon-essential amino acids (SIGMA, Cat.-No. M7145) and 50 μMβ-Mercaptoethanol (SIGMA, M3148).

PBMCs were used directly after isolation (binding on naïve human PBMCs)or they were stimulated to receive a strong human Ox40 expression on thecell surface of T cells (binding on activated human PBMCs). Thereforenaïve PBMCs were cultured for five days in T cell medium supplied with200 U/mL Proleukin and 2 ug/mL PHA-L in 6-well tissue culture plate andthen 1 day on pre-coated 6-well tissue culture plates [2 ug/mLanti-human CD3 (clone OKT3) and 2 ug/mL anti-human CD28 (clone CD28.2)]in T cell medium supplied with 200 U/mL Proleukin at 37° C. and 5% CO₂.

For detection of Ox40 naïve human PBMC and activated human PBMC weremixed. To enable distinction of naïve from activated human PBMC restingcells were labeled prior to the binding assay using the eFluor670 cellproliferation dye (eBioscience, Cat.-No. 65-0840-85).

For labeling cells were harvested, washed with pre-warmed (37° C.) DPBSand adjusted to a cell density of 1×10⁷ cells/mL in DPBS. eFluor670 cellproliferation dye (eBioscience, Cat.-No. 65-0840-85) was added to thesuspension of naïve human PBMC at a final concentration of 2.5 mM and afinal cell density of 0.5×10⁷ cells/mL in DPBS. Cells were thenincubated for 10 min at room temperature in the dark. To stop labelingreaction 2 mL FBS were added and ells were washed three times with Tcell medium. A one to one mixture of 1×10⁵ naïve, eFluor670 labeledhuman PBMC and unlabeled activated human PBMC were then added to eachwell of a round-bottom suspension cell 96-well plates (Greiner bio-one,cellstar, Cat. No. 650185).

Plates were centrifuged 4 minutes with 400×g and at 4° C. andsupernatant was flicked off. Cell were washed once with 200 μL 4° C.cold FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco,Cat. No. E177) and 7.5 mM Sodium azide (Sigma-Aldrich S2002)). Cellswere incubated in 50 μL/well of 4° C. cold FACS buffer containingtitrated anti-Ox40 antibody constructs for 120 minutes at 4° C. Plateswere washed four times with 200 μL/well 4° C. FACS buffer to removeunbound construct.

Cells were stained for 30 minutes at 4° C. in the dark in 25 μL/well 4°C. cold FACS buffer containing fluorescently labeled anti-human CD4(clone RPA-T4, mouse IgG1 k, BioLegend, Cat.-No. 300532), anti-human CD8(clone RPa-T8, mouse IgG1k, BioLegend, Cat.-No. 3010441), anti-humanCD45 (clone HI30, mouse IgG1k, BioLegend, Cat.-No. 304028), andFluorescein isothiocyanate (FITC)-conjugated AffiniPure anti-human IgGFcγ-fragment-specific goat IgG F(ab′)₂ fragment (Jackson ImmunoResearch,Cat.-No. 109-096-098).

Plates where washed twice with 200 μL/well 4° C. FACS buffer, werefinally resuspended in 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI(Santa Cruz Biotec, Cat. No. Sc-3598) and acquired the same day using5-laser LSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 2A-2D, no antibody construct specific for OX40 boundto resting human CD4⁺ T-cells or CD8⁺ T-cells, which do not expressOX40. In contrast, all constructs bound to activated CD8⁺ or CD4⁺T-cells, which do express OX40. Binding to CD4⁺ T-cells was muchstronger than that to CD8⁺ T cells. Activated human CD8⁺ T cells doexpress only a fraction of the OX40 levels detected on activated CD4⁺ Tcells. The difference is donor as well as time dependent. The analyzedanti-OX40 clones varied in their binding strength. The EC₅₀ values areshown in Table 17. For further evaluation of bivalent and monovalent FAPtargeted constructs clones with high (8H9) and low (49B4/1G4) bindingcapacity were chosen.

TABLE 17 EC₅₀ values of binding to activated human CD4 T cells CloneEC₅₀ [nM] 8H9 0.59 CLC563 1.59 20B7 1.64 49B4 4.19 CLC-564 4.63 1G4 n.a.2.2 Binding on Murine OX402.2.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of the phage-derived OX40 specific antibody 20B7 to recombinantmurine OX40 Fc(kih) was assessed by surface plasmon resonance asdescribed above for human OX40 Fc(kih) (see Example 2.1.1). Kineticconstants were derived using the Biacore T200 Evaluation Software (vAA,Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuirbinding by numerical integration and used to estimate qualitatively theavidity (Table 18).

For affinity determination, due to an unspecific interaction of the Fcfusion protein to the reference flow cell, murine Ox40 His (see Example2.1.2) or Ox40 Fc(kih) cleaved with AcTEV protease was used. Anti-humanFc antibody (Biacore, Freiburg/Germany) was directly coupled on a CM5chip at pH 5.0 using the standard amine coupling kit (Biacore,Freiburg/Germany). The immobilization level was approximately 8000 RU.Phage display derived antibodies to OX40 were captured for 60 seconds atconcentrations of 25 nM. Recombinant murine OX40 (cleaved by AcTEVdigestion following the distributor instruction) was passed at aconcentration range from 4.1 to 1000 nM with a flow of 30 μL/minutesthrough the flow cells over 120 seconds. The dissociation was monitoredfor 120 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantigens were flown over a surface with immobilized anti-human Fabantibody but on which HBS-EP has been injected rather than theantibodies.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration. It was shown that clone20B7 binds murine OX40 (Table 18).

Affinity constants of interaction between anti-OX40 P329GLALA IgG1molecules and murine OX40 were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration.

TABLE 18 Binding of anti-Ox40 antibody 20B7 to murine OX40 Recombinantmurine OX40 Recombinant (affinity format) murine OX40 ka kd KD CloneOrigin (avidity format) (1/Ms) (1/s) (M) 20B7 Phage ++ 4.9E+04 1.8E−023.6E−07 display2.2.2 Binding to Mouse OX40 Expressing Cells: Naïve and Activated MouseSplenocytes (Selected Clones)

Mouse spleens were collected in 3 mL PBS and a single cell suspensionwas generated using the gentle MACS tubes (Miltenyi Biotec Cat.-No.130-096-334) and gentleMACS Octo Dissociator (Miltenyi Biotec).Afterwards splenocytes were filtered through a 30 μm pre-separationfilters (Miltenyi Biotec Cat.-No. 130-041-407) and centrifuged for 7 minat 350×g and 4° C. Supernatant was aspirated and cells were resuspendedin RPMI 1640 medium supplied with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 1mM Sodium-Pyruvate, 1% (v/v) MEM non-essential amino acids, 50 μMβ-Mercaptoethanol and 10% Penicillin-Streptomycin (SIGMA, Cat.-No.P4333). 10⁶ cells/mL were cultured for 3 days in a 6-well tissue cultureplate coated with 10 μg/mL anti-mouse CD3ε Armenian hamster IgG (clone145-2C11, BioLegend, Cat.-No. 100331) and 2 μg/mL anti-mouse CD28 Syrianhamster IgG (clone 37.51, BioLegend, Cat.-No. 102102). Activated orfresh mouse splenocytes were harvested, washed in DPBS, counted and0.1×10⁶ cells were transferred to each well of a 96 U-bottom non-tissueculture treated well plate. Cells were washed with DPBS and stained in50 uL FACS buffer containing different concentration of anti-OX40 humanIgG1 P329GLALA antibodies (selected binders only). Cells were incubatedfor 120 min at 4° C. Then cells were washed twice with FACS buffer andstained in 25 FACS buffer containing anti-mouse CD8b rat IgG2bκ-APC-Cy7(BioLegend, Cat.-No. 100714, clone53-6.7), anti-mouse CD4 ratIgG2bκ-PE-Cy7 (BioLegend, Cat.-No. 100422, clone GK1.5) andFITC-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goat IgGF(ab′)₂ fragment (Jackson ImmunoResearch, Cat.-No. 109-096-098) for 30min at 4° C. Plates where washed twice with 200 μL/well 4° C. FACSbuffer, were finally resuspended in 80 μL/well FACS-buffer containing0.2 μg/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) and acquired thesame day using 5-laser LSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 3A-3D only clone 20B7 and the well characterized mousespecific benchmark antibody OX86 showed binding to activated mouse CD4⁺and CD8⁺ T cells. No binding was observed on resting mouse splenocytes.

2.3 Binding on Cynomolgus OX40

To test the reactivity of selected anti-OX40 binders with cynomolguscells, PBMC of healthy Macaca fascicularis were isolated fromheparinized blood using density gradient centrifugation as described forhuman cells with minor modifications. Cynomolgus PBMC were isolated withdensity gradient centrifugation from heparinized fresh blood usinglymphoprep medium (90% v/v, Axon Lab, Cat. No. 1114545) diluted withDPBS. Centrifugation was performed at 520×g, without brake at roomtemperature for 30 minutes. Adjacent centrifugation at 150×g at roomtemperature for 15 minutes was performed to reduce platelets countfollowed by several centrifugation steps with 400×g at room temperaturefor 10 minutes to wash PBMC with sterile DPBS. PBMCs were stimulated toreceive a strong Ox40 expression on the cell surface of T cells (bindingon activated cynomolgus PBMCs). Therefore naïve PBMCs were cultured for72 hrs on pre-coated 12-well tissue culture plates [10 ug/mL cynomolguscross-reactive anti-human CD3 (clone clone SP34)] and 2 ug/mL cynomolguscross-reactive anti-human CD28 (clone CD28.2)] in T cell medium suppliedwith 200 U/mL Proleukin at 37° C. and 5% CO₂.

0.5×10⁵ activated cynomolgus PBMC were then added to each well of around-bottom suspension cell 96-well plates (greiner bio-one, cellstar,Cat. No. 650185). Cell were washed once with 200 μL 4° C. cold FACSbuffer and were incubated in 50 μL/well of 4° C. cold FACS containingtitrated anti-Ox40 antibody constructs for 120 minutes at 4° C. Then,plates were washed four times with 200 μL/well 4° C. FACS buffer. Cellswere resuspended in 25 μL/well 4° C. cold FACS buffer containingfluorescently labeled, cynomolgus cross-reactive anti-human CD4 (cloneOKT-4, mouse IgG1 k, BD, Cat.-No. 317428), anti-human CD8 (clone HIT8a,mouse IgG1k, BD, Cat.-No. 555369) and FITC-conjugated AffiniPureanti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂ fragment (JacksonImmunoResearch, Cat.-No. 109-096-098) and incubated for 30 minutes at 4°C. in the dark. Plates where washed twice with 200 μL/well 4° C. FACSbuffer, were finally resuspended in 80 μL/well FACS-buffer containing0.2 μg/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) and acquired thesame day using 5-laser LSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 4A and 4B, most constructs bound to activated CD4⁺cynomolgus T-cells. Binding to CD4⁺ T-cells was much stronger than thatto CD8⁺ T cells. Expression levels for OX40 are depending on kinetic andstrength of stimulation and were optimized for CD4⁺ cynomolgus T cellsbut not for CD8⁺ cynomolgus T cells, so that only little OX40 expressionwas induced on CD8⁺ T cells. The analyzed anti-OX40 clones varied intheir binding strength. The EC₅₀ values are shown in Table 19. Due tountypical curve fit no EC₅₀ value could be calculated for clones 8H9,49B4, 21H4.

TABLE 19 EC₅₀ values of binding to activated cynomolgus CD4 T cellsClone EC₅₀ [nM] 8H9 n.d. CLC563 1.41 20B7 1.52 49B4 n.d. CLC-564 3.501G4 48.20 2.3.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of phage-derived OX40-specific antibodies (all human IgG1P329GLALA) to the recombinant cynomolgus OX40 Fc(kih) was assessed bysurface plasmon resonance (SPR). All SPR experiments were performed on aBiacore T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,Freiburg/Germany).

Biotinylated cynomolgus OX40 Fc(kih) was directly coupled to differentflow cells of a streptavidin (SA) sensor chip. Immobilization levels upto 800 resonance units (RU) were used.

Phage display derived anti-OX40 human IgG1 P329GLALA antibodies werepassed at a concentration range from 2 to 500 nM (3-fold dilution) witha flow of 30 μL/minute through the flow cells over 120 seconds. Complexdissociation was monitored for 210 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained in areference flow cell, where no protein was immobilized.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration and used to estimatequalitatively the avidity (Table 20).

In the same experiment, the affinities of the interaction between phagedisplay derived antibodies (human IgG1 P329GLALA) to recombinantcynomolgus OX40 Fc(kih) were determined. Anti-human Fab antibody(Biacore, Freiburg/Germany) was directly coupled on a CM5 chip at pH 5.0using the standard amine coupling kit (Biacore, Freiburg/Germany). Theimmobilization level was approximately 9000 RU. Phage display derivedantibodies to Ox40 were captured for 60 seconds at concentrations of 25to 50 nM. Recombinant cynomolgus Ox40 Fc(kih) was passed at aconcentration range from 4 to 1000 nM with a flow of 30 μL/minutesthrough the flow cells over 120 seconds. The dissociation was monitoredfor 120 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantigens were flown over a surface with immobilized anti-human Fabantibody but on which HBS-EP has been injected rather than theantibodies.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration (Table 20).

Clones 49B4, 1G4 and CLC-564 bind cynomolgus OX40 Fc(kih) with a loweraffinity than clones 8H9, 20B7 and CLC-563.

Affinity constants of interaction between anti-OX40 P329GLALA IgG1 andcynomolgus OX40 Fc(kih) were derived using the Biacore T100 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration.

TABLE 20 Binding of anti-OX40 antibodies to recombinant cynomolgus OX40Fc(kih) Recombinant Recombinant cynomolgus OX40 cynomolgus (affinityformat) OX40 (avidity ka kd KD Clone Origin format) (1/Ms) (1/s) (M) 8H9Phage ++++ 1.4E+05 9.6E−02 6.7E−07 display 20B7 Phage +++ 1.57E+04 1.66E−02  1.1E−06 display 49B4 Phage ++ 1.1E+05 3.8E−02 3.5E−07 display1G4 Phage + Too low to be detected display CLC- Phage +++ 2.8E+046.9E−04 2.5E−08 563 display CLC- Phage +++ 2.1E+04 7.2E−04 3.4E−08 564display2.3.2 Binding on Cynomolgus OX40 Expressing Cells: Activated CynomolgusPeripheral Mononuclear Blood Leukocytes (PBMC)Binding to OX40 Negative Tumor Cells

The lack of binding to OX40 negative tumor cells was tested usingWM266-4 cells (ATCC CRL-1676) and U-87 MG (ATCC HTB-14) tumor cells. Toallow separation of both tumor cells, WM266-4 cells were pre-labeledwith PKH-26 Red Fluorescence Cell linker Kit (Sigma, Cat.-No. PKH26GL).Cells were harvested and washed three times with RPMI 1640 medium.Pellet was stained for 5 minutes at room temperature in the dark at afinal cell density of 1×10⁷ cells in freshly prepared PKH26-Red-stainsolution (final concentration [1 nM] in provided diluent C). Excess FBSwas added to stop labeling reaction and cell were washed four times withRPMI 1640 medium supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I toremove excess dye.

A mixture of 5×10⁴ PKH26 labeled WM266-4 cells and unlabeled U-87 MGcells in DPBS were added to each well of a round-bottom suspension cell96-well plates. Plates were centrifuged 4 minutes, 400×g, 4° C. andsupernatant were flicked off. Cells were washed once with 200 μL DPBSand pellets were resuspended by a short and gentle vortex. All sampleswere resuspended in 50 μL/well of 4° C. cold FACS buffer containingtitrated concentrations of anti-Ox40 human IgG1 P329GLALA antibodyconstructs for 120 minutes at 4° C. Plates were washed four times with200 μL/well 4° C. FACS buffer. Cells were resuspended in 25 μL/well 4°C. cold FACS buffer containing FITC-conjugated AffiniPure anti-human IgGFcγ-fragment-specific goat IgG F(ab′)₂ fragment (Jackson ImmunoResearch,Cat.-No. 109-096-098) and incubated for 30 minutes at 4° C. in the dark.Plates where washed twice with 200 μL/well 4° C. FACS buffer, werefinally resuspended in 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI(Santa Cruz Biotec, Cat. No. Sc-3598) and acquired the same day using5-laser LSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 5A and 5B, no antibody construct specific for OX40bound to OX40 negative human tumor cells WM266-4 and U-78 MG.

2.4 Ligand Blocking Property

To determine the capacity of OX40-specific human IgG1 P329GLALA antibodymolecules to interfere with OX40/OX40-ligand interactions human OX40ligand (R&D systems) was used. Due to the low affinity of theinteraction between OX40 and OX40 ligand, a dimeric human OX40 Fc fusionwith a C-terminal Ha tag was prepared (FIG. 1B). The nucleotide andamino acid sequences of this dimeric human Ox40 Fc fusion molecule areshown in Table 21. Production and purification were performed asdescribed for the monomeric OX40 Fc(kih) in Example 1.1.

TABLE 21 cDNA and Amino acid sequences of dimeric human OX40 Fc fusionmolecule (composed by 2 Fc chains) SEQ ID NO: Antigen Sequence 212Nucleotide CTGCACTGCGTGGGCGACACCTACCCCAGCAACGACC sequenceGGTGCTGCCACGAGTGCAGACCCGGCAACGGCATGGT dimeric humanGTCCCGGTGCAGCCGGTCCCAGAACACCGTGTGCAGA OX40 antigenCCTTGCGGCCCTGGCTTCTACAACGACGTGGTGTCCAG FcCAAGCCCTGCAAGCCTTGTACCTGGTGCAACCTGCGGAGCGGCAGCGAGCGGAAGCAGCTGTGTACCGCCACCCAGGATACCGTGTGCCGGTGTAGAGCCGGCACCCAGCCCCTGGACAGCTACAAACCCGGCGTGGACTGCGCCCCTTGCCCTCCTGGCCACTTCAGCCCTGGCGACAACCAGGCCTGCAAGCCTTGGACCAACTGCACCCTGGCCGGCAAGCACACCCTGCAGCCCGCCAGCAATAGCAGCGACGCCATCTGCGAGGACCGGGATCCTCCTGCCACCCAGCCTCAGGAAACCCAGGGCCCTCCCGCCAGACCCATCACCGTGCAGCCTACAGAGGCCTGGCCCAGAACCAGCCAGGGGCCTAGCACCAGACCCGTGGAAGTGCCTGGCGGCAGAGCCGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGCTACCCATACG ATGTTCCAGATTACGCT 213 dimerichuman LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRP OX40 antigenCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQD FcTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSGYPYDVPDYA

Human OX40 ligand (R&D systems) was directly coupled to two flow cellsof a CM5 chip at approximately 2500 RU by pH 5.0 using the standardamine coupling kit (Biacore, Freiburg/Germany). Recombinant human Ox40Fc was passed on the second flow cell at a concentration of 200 nM witha flow of 30 μL/minute over 90 seconds. The dissociation was omitted andthe phage derived anti-Ox40 human IgG1P329LALA was passed on both flowcells at a concentration of 500 nM with a flow of 30 μL/minute over 90seconds. The dissociation was monitored for 60 seconds. Bulk refractiveindex differences were corrected for by subtracting the responseobtained on reference flow cell. Here, the antibodies were flown over asurface with immobilized human OX40 ligand but on which HBS-EP has beeninjected instead of recombinant human OX40 Fc. FIG. 1C shows the designof the experiment.

The phage-derived clone 20B7 bound to the complex of human OX40 with itsOX40 ligand (Table 22, FIG. 6B). Thus, this antibody does not competewith the ligand for binding to human OX40 and is therefore termed“non-ligand blocking”. On the contrary, clones 8H9, 1G4, 49B4, CLC-563and CLC-564 did not bind to human OX40 in complex with its ligand andare therefore termed “ligand blocking”.

TABLE 22 Ligand binding property of the anti-OX40 clones determined bysurface plasmon resonance Second injection (anti- Ligand Clone OriginFirst injection Ox40 clone) blocking 8H9 Phage human OX40 Not bindingYES display Fc 20B7 Phage human OX40 Binding NO display Fc 1G4 Phagehuman OX40 Not binding YES display Fc 49B4 Phage human OX40 Not bindingYES display Fc CLC-564 Phage human OX40 Not binding YES display FcCLC-564 Phage human OX40 Not binding YES display Fc

Example 3 Functional Properties of Anti-human OX40 Binding Clones

3.1 HeLa Cells Expressing Human OX40 and Reporter Gene NF-κB-luciferase

Agonstic binding of OX40 to its ligand induces downstream signaling viaactivation of nuclear factor kappa B (NFκB) (A. D. Weinberg et al., J.Leukoc. Biol. 2004, 75(6), 962-972). The recombinant reporter cell lineHeLa_hOx40_NFkB_Luc1 was generated to express human Ox40 on its surface.Additionally, it harbors a reporter plasmid containing the luciferasegene under the control of an NFκB-sensitive enhancer segment. Ox40triggering induces dose-dependent activation of NFκB, which translocatesin the nucleus, where it binds on the NFκB sensitive enhancer of thereporter plasmid to increase expression of the luciferase protein.Luciferase catalyzes luciferin-oxidation resulting in oxyluciferin whichemits light. This can be quantified by a luminometer. The scope of oneexperiment was to test the capacity of the various anti-Ox40 binders ina P329GLALA huIgG1 format to induce NFκB activation inHeLa_hOx40_NFκB_Luc1 reporter cells.

Adherent HeLa_hOx40_NFκB_Luc1 cells were harvested using celldissociation buffer (Invitrogen, Cat.-No. 13151-014) for 10 minutes at37° C. Cells were washed once with DPBS and were adjusted to a celldensity of 2×10⁵ in assay media comprising of MEM (Invitrogen, Cat.-No.22561-021), 10% (v/v) heat-inactivated FBS, 1 mM Sodium-Pyruvat and 1%(v/v) non-essential amino acids. Cells were seeded in a density of0.3*10⁵ cells per well in a sterile white 96-well flat bottom tissueculture plate with lid (greiner bio-one, Cat. No. 655083) and kept overnight at 37° C. and 5% CO₂ in an incubator (Hera Cell 150).

The next day, HeLa_hOX40_NFkB_Luc1 were stimulated for 6 hours addingassay medium containing various titrated anti-OX40 binders in aP329GLALA huIgG1 format. For testing the effect of hyper-crosslinking onanti-OX40 antibodies, 50 μL/well of medium containing secondary antibodyanti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂ fragment (JacksonImmunoResearch, 109-006-098) were added in a 1:2 ratio (2 times moresecondary antibody than the primary single anti-OX40 P329GLALA huIgG1).After incubation, supernatant was aspirated and plates washed two timeswith DPBS. Quantification of light emission was done using theluciferase 1000 assay system and the reporter lysis buffer (bothPromega, Cat.-No. E4550 and Cat-No: E3971) according to manufacturerinstructions. Briefly, cells were lysed for 10 minutes at −20° C. byaddition of 30 uL per well 1× lysis buffer. Cells were thawed for 20minutes at 37° C. before 90 uL per well provided luciferase assayreagent was added. Light emission was quantified immediately with aSpectraMax M5/M5e microplate reader (Molecular Devices, USA) using 500ms integration time, without any filter to collect all wavelengths.Emitted relative light units (URL) were corrected by basal luminescenceof HeLa_hOX40_NFκB_Luc1 cells and were blotted against the logarithmicprimary antibody concentration using Prism4 (GraphPad Software, USA).Curves were fitted using the inbuilt sigmoidal dose response.

As shown in FIGS. 7A and 7B, a limited, dose dependent NFκB activationwas induced already by addition of anti-OX40 P329GLALA huIgG1 antibodies(left side) to the reporter cell line. Hyper-crosslinking of anti-OX40antibodies by anti-human IgG specific secondary antibodies stronglyincreased the induction of NFκB-mediated luciferase-activation in aconcentration-dependent manner (right side). The EC₅₀ values ofactivation are summarized in Table 23.

TABLE 23 EC₅₀ values of NFκB activation in the HeLa_hOx40_NFκB_luc1reporter cell line co-incubated with anti-Ox40 binders (huIgG1 P329GLALAformat) and secondary anti-human IgG Fcγ spec. antibodies Clone EC₅₀[nM] 8H9 0.66 CLC563 1.69 20B7 2.27 49B4 2.42 CLC-564 3.23 1G4 3.593.2 OX40 Mediated Costimulation of Suboptimally TCR TriggeredPre-activated Human CD4 T Cells

Ligation of OX40 provides a synergistic co-stimulatory signal promotingdivision and survival of T-cells following suboptimal T-cell receptor(TCR) stimulation (M. Croft et al., Immunol. Rev. 2009, 229(1),173-191). Additionally, production of several cytokines and surfaceexpression of T-cell activation markers is increased (I. Gramaglia etal., J. Immunol. 1998, 161(12), 6510-6517; S. M. Jensen et al., Seminarsin Oncology 2010, 37(5), 524-532).

To test agonistic properties of various anti-OX40 binders, pre-activatedOx40 positive CD4 T-cells were stimulated for 72 hours with a suboptimalconcentration of plate-immobilized anti-CD3 antibodies in the presenceof anti-OX40 antibodies, either in solution or immobilized on the platesurface. Effects on T-cell survival and proliferation were analyzedthrough monitoring of total cell counts and CFSE dilution in livingcells by flow cytometry. Additionally, cells were co-stained withfluorescently-labeled antibodies against T-cell activation anddifferentiation markers, e.g. CD127, CD45RA, Tim-3, CD62L and OX40itself.

Human PBMCs were isolated via ficoll density centrifugation and weresimulated for three days with PHA-L [2 μg/mL] and Proleukin [200 U/mL]as described under Example 2.1.2. Cells were then labeled with CFSE at acell density of 1×10⁶ cells/mL with CFDA-SE (Sigma-Aldrich, Cat.-No.2188) at a final concentration of [50 nM] for 10 minutes at 37° C.Thereafter, cells were washed twice with excess DPBS containing FBS (10%v/v). Labeled cells were rested in T-cell media at 37° C. for 30minutes. Thereafter, non-converted CFDA-SE was removed by two additionalwashing steps with DPBS. CD4 T-cell isolation from pre-activatedCFSE-labeled human PBMC was performed using the MACS negative CD4 T-cellisolation kit (Miltenyi Biotec,) according to manufacturer instructions.

Morris et al. showed that agonistic co-stimulation with conventionalanti-Ox40 antibodies relied on surface immobilization (N. P. Morris etal., Mol. Immunol. 2007, 44(12), 3112-3121). Thus, goat anti-mouseFcγ-specific antibodies (Jackson ImmunoResearch, Cat.No. 111-500-5008)were coated to the surface of a 96 well U-bottom cell culture plate(Greiner Bio One) at a concentration of [2 μg/mL] in PBS over night at4° C. in the presence (surface immobilized anti-OX40) or absence(anti-OX40 in solution) of goat anti-human Fcγ-specific antibody(Jackson ImmunoResearch, Ca.No. 109-006-098). Thereafter, the platesurface was blocked with DPBS containing BSA (1% v/w). All T cellfollowing incubation steps were done at 37° C. for 90 minutes in PBScontaining BSA (1% v/w). Between the incubation steps, plates werewashed with DPBS.

Mouse anti-human CD3 antibody (clone OKT3, eBioscience, Ca.No.16-0037-85, fixed concentration [3 ng/mL]) was captured in a subsequentincubation step via the surface coated anti-mouse Fcγ-specificantibodies. In one experiment titrated human anti-OX40 antibodies (humanIgG₁ P329G LALA) were then immobilized on plate by an additionalincubation step in DPBS. In a second experiment anti-OX40 antibodieswere added during the activation assay directly to the media to platesnot pre-coated with anti-human IgG Fc specific antibodies.

CFSE-labeled preactivated CD4⁺ T cells were added to the pre-coatedplates at a cell density of 0.6*10⁵ cells per well in 200 μL T-cellmedia and cultured for 96 hours. Cells were stained with a combinationof fluorochrome-labeled mouse anti-human Ox40 (clone BerACT35,Bioledgend, Ca.No. 35008), TIM-3 (clone F38-2E2, Biolegend, Ca.No.345008), CD127 (clone A019D5, Biolegend, Ca.No. 351234), CD62L (cloneDREG 56, Biolegend, Ca.No. 304834) and CD45RA (clone HI100, BDBiosciences, Ca.No. 555489) for 20 minutes at 4° C. in the dark. Plateswhere washed twice with 200 μL/well 4° C. FACS buffer, were finallyresuspended in 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI (SantaCruz Biotec, Cat. No. Sc-3598) and acquired the same day using 5-laserLSR-Fortessa (BD Bioscience with DIVA software).

DAPI negative living cells were analyzed for decrease in median CFSEfluorescence as a marker for proliferation. The percentage of OX40positive, CD62L low and TIM-3 positive T cells was monitored as a markerfor T-cell activation. The expression of CD45RA and CD127 was analyzedto determine changes in maturation status of T cell, whereby CD45RA lowCD127 low cells were categorized as effector T cells.

Co-stimulation with plate-immobilized antibodies strongly enhancedsuboptimal stimulation of pre-activated human CD4 T cells withplate-immobilized anti-human CD3 in a dose dependent manner (FIGS.8A-8F). T-cells proliferated stronger, showed a more mature phenotypewith a higher percentage of effector T cells and had higher percentagesof CD62L low, Tim-3 positive and OX40 positive activated cells. Someclones (8H9, 20B7) out-competed the commercially available detectionantibody in binding to cellular OX40. For those no EC₅₀ valuecalculation was possible and thus all EC₅₀ values for OX40 inductionwere excluded from overall EC₅₀ value calculation. Half-maximal changesin all other parameters of T-cell activation were achieved atconcentrations ranging from 3 to 700 pM and are summarized in FIG. 9 andTable 24. No enhancement in suboptimal TCR stimulation was seen whenanti-Ox40 antibodies were added in solution in the absence of surfaceimmobilization (FIGS. 10A-10F). This demonstrated again the strongdependency of Ox40 axis activation on hypercrosslinking of the OX40receptor.

A correlation between the binding strength and the agonistic activity(bioactivity) of the anti-OX40 antibodies (hu IgG1 P329GLALA format) isshown in FIG. 11. For most clones there was a direct correlation,however surprisingly two clones (49B4, 1G4) showed a much strongerbioactivity then was predicted from their binding strength.

TABLE 24 EC₅₀ values of of rescuing suboptimal TCR stimulation withplate- immobilized anti-OX40 binders (huIgG1 P329GLALA format) CloneEC₅₀ [nM] SEM (+/−) 8H9 0.003 0.001 20B7 0.090 0.015 CLC-563 0.114 0.018CLC-564 0.202 0.053 49B4 0.591 0.237 1G4 0.697 0.278

Example 4 Generation of Bispecific Constructs Targeting Ox40 andFibroblast Activation Protein (FAP)

4.1 Generation of Bispecific Bivalent Antigen Binding MoleculesTargeting Ox40 and Fibroblast Activation Protein (FAP) (2+2 Format)

Bispecific agonistic Ox40 antibodies with bivalent binding for Ox40 andfor FAP were prepared. The crossmab technology in accordance withInternational patent application No. WO 2010/145792 A1 was applied toreduce the formation of wrongly paired light chains.

The generation and preparation of the FAP binders is described in WO2012/020006 A2, which is incorporated herein by reference.

In this example, a crossed Fab unit (VHCL) of the FAP binder 28H1 wasC-terminally fused to the heavy chain of an anti-OX40 huIgG1 using a(G4S)₄ connector sequence. This heavy chain fusion was co-expressed withthe light chain of the anti-OX40 and the corresponding FAP crossed lightchain (VLCH1). The Pro329Gly, Leu234Ala and Leu235Ala mutations havebeen introduced in the constant region of the heavy chains to abrogatebinding to Fc gamma receptors according to the method described inInternational Patent Appl. Publ. No. WO 2012/130831 A1. The resultingbispecific, bivalent construct is depicted in FIG. 12A.

Table 25 shows, respectively, the nucleotide and amino acid sequences ofmature bispecific, bivalent anti-OX40/anti-FAP human IgG1 P329GLALAantibodies.

TABLE 25 Sequences of bispecific, bivalent anti-OX40/anti-FAP human IgG1P329GLALA antigen binding molecules SEQ ID NO: Description Sequence 214(8B9) VHCH1- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTGGATGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGG GCGAGTGC 180 VLCL-Light seeTable 13 chain 1 (8B9) (nucleotide sequence) 215 VLCH1-LightGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGA chain 2 (28H1)GCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCC (nucleotideAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGA sequence)AGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGAGCTCCGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCCAGCAGCAAGAGCACCTCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCC AAGAGCTGCGAC 216 (8B9) VHCH1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 182 VLCL-Light see Table 13 chain 1(8B9) 217 VLCH1-Light EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPG chain 2(28H1) QAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CD 218 (49B4) VHCH1-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC CGGGGCGAGTGC 184 VLCL-Light seeTable 13 chain 1 (49B4) (nucleotide sequence) 215 VLCH1-Light see abovechain 2 (28H1) (nucleotide sequence) 219 (49B4) VHCH1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 186 VLCL-Light see Table 13 chain 1(49B4) 217 VLCH1-Light see above chain 2 (28H1) 220 (1G4) VHCH1-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTCTATGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGG GCGAGTGC 188 VLCL-Light seeTable 13 chain 1 (1G4) (nucleotide sequence) 215 VLCH1-Light see abovechain 2 (28H1) (nucleotide sequence) 221 (1G4) VHCH1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCAREYGSMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 190 VLCL-Light see Table 13 chain 1(1G4) 217 VLCH1-Light see above chain 2 (28H1) 222 (20B7) VHCH1-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGTTAACTACCCGTACTCTTACTGGGGTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCG TGACCAAGTCCTTCAACCGGGGCGAGTGC192 VLCL-Light see Table 13 chain 1 (20B7) (nucleotide sequence) 215VLCH1-Light see above chain 2 (28H1) (nucleotide sequence) 223 (20B7)VHCH1- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCARVNYPYSYWGDFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 194 VLCL-Light see Table 13chain 1 (20B7) 217 VLCH1-Light see above chain 2 (28H1) 224 (CLC-563)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG VHCH1-HeavyCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT chain-(28H1)TCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC VHCLTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGG (nucleotideTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGG sequence)CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCTTGACGTTGGTGCTTTCGACTACTGGGGCCAAGGAGCCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGG GCGAGTGC 196 VLCL-Light seeTable 13 chain 1 (CLC- 563) (nucleotide sequence) 215 VLCH1-Light seeabove chain 2 (28H1) (nucleotide sequence) 225 (CLC-563)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP VHCH1-HeavyGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ chain-(28H1)MNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKG VHCLPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 198 VLCL-Light see Table 13 chain 1(CLC- 563) 217 VLCH1-Light see above chain 2 (28H1) 226 (CLC-564)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG VHCH1-HeavyCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT chain-(28H1)TCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC VHCLTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGG (nucleotideTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGG sequence)CCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGTTCGACGTTGGTCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGG GCGAGTGC 200 VLCL-Light seeTable 13 chain 1 (CLC- 564) (nucleotide sequence) 215 VLCH1-Light seeabove chain 2 (28H1) (nucleotide sequence) 227 (CLC-564)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP VHCH1-HeavyGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ chain-(28H1)MNSLRAEDTAVYYCAFDVGPFDYWGQGTLVTVSSASTKGP VHCLSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 202 VLCL-Light see Table 13 chain 1(CLC- 564) 217 VLCH1-Light see above chain 2 (28H1)

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

The bispecific anti-Ox40, anti-FAP constructs were produced byco-transfecting HEK293-EBNA cells with the mammalian expression vectorsusing polyethylenimine. The cells were transfected with thecorresponding expression vectors in a 1:1:1 ratio (“vector heavychain”:“vector light chain1”:“vector light chain2”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes by 210×g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO₂ atmosphere. After theincubation, 160 mL F17 medium was added and cells were cultured for 24hours. One day after transfection 1 mM valproic acid and 7% Feed wereadded. After culturing for 7 days, the cell supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of bispecific constructs from cell culture supernatants wascarried out by affinity chromatography using Protein A as describedabove for purification of antigen-Fc fusions and antibodies.

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl solution of pH 6.0.

The protein concentration of purified bispecific constructs wasdetermined by measuring the OD at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of the bispecific constructs were analyzed byCE-SDS in the presence and absence of a reducing agent (Invitrogen, USA)using a LabChipGXII (Caliper). The aggregate content of bispecificconstructs was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in a 25 mM K2HPO4, 125 mMNaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7running buffer at 25° C. (Table 26).

TABLE 26 Biochemical analysis of exemplary bispecific, bivalent anti-Ox40/anti-FAP IgG1 P329G LALA antigen binding molecules Yield MonomerClone [mg/l] [%] CE-SDS (non red) CE-SDS (red) 8H9/FAP P329GLALA 58 10095.3% (254 kDa) 3.2% (114 kDa) IgG1 2 + 2 3% (237 kDa) 71.3% (90.7 kDa)13.3% (28.9 kDa) 11.9% (26.2 kDa) 49B4/FAP P329GLALA 17 99 98.9% (253kDa) 3.% (116 kDa) IgG1 2 + 2 71.4% (92 kDa) 12.9% (28.9 kDa) 12.1%(25.7 kDa) 1G4/FAP P329GLALA 0.5 99.1 93.9% (234 kDa) 55.5% (90.6 kDa)IgG1 2 + 2 3.2% (242 kDa) 20.7% (27 kDa) 1.2% (244 kDa) 21.6% (25 kDa)20B7/FAP P329GLALA 14 97.2 91.5% (244 kDa) 54.1% (89 kDa) IgG1 2 + 22.3% (227 kDa) 19% (27 kDa) 1.4% (218 kDa) 25% (24 kDa) 1.5% (202 kDa)4.2 Generation of Bispecific Monovalent Antigen Binding MoleculesTargeting Ox40 and Fibroblast Activation Protein (FAP) (1+1 Format)

Bispecific agonistic Ox40 antibodies with monovalent binding for Ox40and for FAP were prepared by applying the crossmab technology accordingto International patent application No. WO 2010/145792 A1 to reduce theformation of wrongly paired light chains.

In this example, a crossed Fab unit (VHCL) of the FAP binder 28H1 wasfused to the hole heavy chain of a huIgG1. The Fab against anti-Ox40 wasfused to the knob heavy chain. Combination of the targeted anti-FAP-Fchole with the anti-Ox40-Fc knob chain allows generation of aheterodimer, which includes a FAP binding Fab and an Ox40 binding Fab(FIG. 12B).

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the heavy chains to abrogate binding to Fc gammareceptors according to the method described in International PatentAppl. Publ. No. WO 2012/130831 A1.

The resulting bispecific, monovalent construct is depicted in FIG. 12Band the nucleotide and amino acid sequences can be found in Table 27.

TABLE 27 cDNA and amino acid sequences of mature bispecific monovalentanti-Ox40/anti-FAP huIgG1 P329GLALA kih antibodies SEQ ID NO:Description Sequence 228 (28H1) VHCL-heavyGAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGC chain holeAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCC (nucleotide sequence)GGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCCAGCGACGAGCAGCTGAAGTCCGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 215 (28H1) VLCH1-Light see Table25 chain 2 (nucleotide sequence) 229 (28H1) VHCL-heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQ chain holeAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 217 (28H1)VLCH1-Light see Table 25 chain 2 302 (8H9) VHCH1-heavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTGGATGGACTACTGGGGCCAAGGGACCACCGTGA CCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 180 (8H9)VLCL-Light see Table 13 chain 1 (nucleotide sequence) 303 (8H9)VHCH1-heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGWMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 182 (8H9)VLCL-Light see Table 13 chain 1 230 (49B4) VHCH1-heavyAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAA chain knobGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCG (nucleotide sequence)GAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCAC CGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 184 (49B4)VLCL-Light see Table 13 chain 1 (nucleotide sequence) 231 (49B4)VHCH1-heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 186 (49B4)VLCL-Light see Table 13 chain 1 232 (1G4) VHCH1-heavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACGGTTCTATGGACTACTGGGGCCAAGGGACCACCGTGA CCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 188 (1G4)VLCL-Light see Table 13 chain 1 (nucleotide sequence) 233 (1G4)VHCH1-heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREYGSMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 190 (1G4)VLCL-Light see Table 13 chain 1 234 (20B7) VHCH1-heavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGTTAACTACCCGTACTCTTACTGGGGTGACTTCGACTACTGGGG CCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 192 (20B7)VLCL-Light see Table 13 chain 1 (nucleotide sequence) 235 (20B7)VHCH1-heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARVNYPYSYWGDFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 194 (20B7)VLCL-Light see Table 13 chain 1 236 (CLC-563) VHCH1-GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC heavy chain knobAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC (nucleotide sequence)GGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCTTGACGTTGGTGCTTTCGACTACTGGGGCCAAGGAGCCCTGGTCACC GTCTCGAGTGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 196 (CLC-563)VLCL- see Table 13 Light chain 1 (nucleotide sequence) 237 (CLC-563)VHCH1- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ heavy chain knobAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALDVGAFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 198 (CLC-563)VLCL- see Table 13 Light chain 1 238 (CLC-564) VHCH1-GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC heavy chain knobAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC (nucleotide sequence)GGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGTTCGACGTTGGTCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACC GTCTCGAGTGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 200 (CLC-564)VLCL- see Table 13 Light chain 1 (nucleotide sequence) 239 (CLC-564)VHCH1- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ heavy chain knobAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAFDVGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 202 (CLC-564)VLCL- see Table 13 Light chain 1

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

The bispecific anti-Ox40, anti-FAP constructs were produced byco-transfecting HEK293-EBNA cells with the mammalian expression vectorsusing polyethylenimine. The cells were transfected with thecorresponding expression vectors in a 1:1:1:1 ratio (“vector heavy chainhole”: “vector heavy chain knob”:“vector light chain1”: “vector lightchain2”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes by 210×g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO₂ atmosphere. After theincubation, 160 mL F17 medium was added and cells were cultured for 24hours. One day after transfection 1 mM valproic acid and 7% Feed wereadded. After culturing for 7 days, the cell supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of the bispecific antigen binding molecules from cellculture supernatants was carried out by affinity chromatography usingProtein A as described above for purification of antigen-Fc fusions andantibodies.

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl solution of pH 6.0.

The protein concentration of purified bispecific constructs wasdetermined by measuring the OD at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of the bispecific constructs were analyzed byCE-SDS in the presence and absence of a reducing agent (Invitrogen, USA)using a LabChipGXII (Caliper). The aggregate content of bispecificconstructs was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in a 25 mM K₂HPO₄, 125 mMNaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7running buffer at 25° C.

Table 28 summarizes the biochemical analysis of bispecific, monovalentanti-Ox40/anti-FAP IgG1 P329G LALA kih antigen binding molecules.

TABLE 28 Biochemical analysis of bispecific monovalent anti-Ox40/anti-FAP IgG1 P329G LALA kih antigen binding molecules Yield Monomer Clone[mg/l] [%] CE-SDS (non red) CE-SDS (red) 8H9/FAP P329GLALA 16.5 10092.1% (164 kDa) 67.7% (63.6 kDa) IgG1 1 + 1 1.9% (145 kDa) 13.3% (28.5kDa) 3.6% (120.1 kDa) 16.5% (25.7 kDa) 1G4/FAP P329GLALA 12.5 98.5 85.2%(157 kDa) 69.5% (64.2 kDa) IgG1 1 + 1 7.4% (151 kDa) 13.1% (28.8 kDa)2.8% (139.5 kDa) 16.7% (26.2 kDa) 49B4/FAP P329GLALA 2.3 97.9 80% (153kDa) 70.4% (63.5 kDa) IgG1 1 + 1 11.9% (141 kDa) 14.7% (28 kDa) 4.3%(120 kDa) 13.7% (25 kDa) 20B7/FAP P329GLALA 22 100 97.5% (166 kDa) 82.7%(56.2 kDa) IgG1 1 + 1 1.3% (149 kDa) 8.2% (27.2 kDa) 8.1% (24.3 kDa)4.3 Characterization of Bispecific Constructs Targeting Ox40 and FAP4.3.1 Surface Plasmon Resonance (Simultaneous Binding)

The capacity of binding simultaneously human Ox40 Fc(kih) and human FAPwas assessed by surface plasmon resonance (SPR). All SPR experimentswere performed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany). Biotinylated human Ox40 Fc(kih) was directlycoupled to a flow cell of a streptavidin (SA) sensor chip.Immobilization levels up to 1000 resonance units (RU) were used.

The bispecific constructs targeting Ox40 and FAP were passed at aconcentration range of 250 nM with a flow of 30 μL/minute through theflow cells over 90 seconds and dissociation was set to zero sec. HumanFAP was injected as second analyte with a flow of 30 μL/minute throughthe flow cells over 90 seconds at a concentration of 250 nM (FIG. 12C).The dissociation was monitored for 120 sec. Bulk refractive indexdifferences were corrected for by subtracting the response obtained in areference flow cell, where no protein was immobilized.

As can be seen in the graphs of FIGS. 13A-13H, all bispecific constructscould bind simultaneously human Ox40 and human FAP.

4.3.2 Binding on Cells

4.3.2.1 Binding to Naïve Versus Activated Human PBMCs of FAP-targetedAnti-Ox40 Antibodies

Human PBMC were isolated by ficoll density gradient centrifugation asdescribed in Example 2.1.2. PBMCs were used directly after isolation(binding on resting human PBMCs) or they were stimulated to receive astrong human Ox40 expression on the cell surface of T cells (binding onactivated human PBMCs). Therefore naïve PBMCs were cultured for four toseven days in T cell medium supplied with 200 U/mL Proleukin and 2 ug/mLPHA-L in 6-well tissue culture plate and then 1 day on pre-coated 6-welltissue culture plates [10 ug/mL anti-human CD3 (clone OKT3) and 2 ug/mLanti-human CD28 (clone CD28.2)] in T cell medium supplied with 200 U/mLProleukin at 37° C. and 5% CO₂.

For detection of Ox40 naïve human PBMC and activated human PBMC weremixed. To enable distinction of naïve from activated human PBMC naïvecells were labeled prior to the binding assay using the eFluor670 cellproliferation dye (eBioscience, Cat.-No. 65-0840-85). A 1 to 1 mixtureof 1×10⁵ naïve, eFluor670 labeled human PBMC and unlabeled activatedhuman PBMC were then added to each well of a round-bottom suspensioncell 96-well plates (greiner bio-one, cellstar, Cat. No. 650185) and thebinding assay was performed as described in Example 2.1.2. A 1 to 1mixture of 1×105 naïve, eFluor670 labeled human PBMC and unlabeledactivated human PBMC were then added to each well of a round-bottomsuspension cell 96-well plates (greiner bio-one, cellstar, Cat. No.650185) and binding assay was performed as described in section 2.1.2.

Primary antibodies were titrated anti-Ox40 antibody constructs,incubated for 120 minutes at 4° C. Secondary antibody solution was amixture of fluorescently labeled anti-human CD4 (clone RPA-T4, mouseIgG1 k, BioLegend, Cat.-No. 300532), anti-human CD8 (clone RPa-T8, mouseIgG1k, BioLegend, Cat.-No. 3010441) and Fluorescein isothiocyanate(FITC)-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatIgG F(ab′)₂ fragment (Jackson ImmunoResearch, Cat.-No. 109-096-098),incubated for 30 minutes at 4° C. in the dark. Plates were finallyresuspended in 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI (SantaCruz Biotec, Cat. No. Sc-3598) and acquired the same day using 5-laserLSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 14A-14Q, no antigen binding molecule specific for Ox40bound to resting human CD4⁺ T-cells or CD8⁺ T-cells. In contrast, allantigen binding molecules bound to activated CD8⁺ or CD4⁺ T-cells.Binding to CD4⁺ T-cells was much stronger than that to CD8⁺ T cellssimilar to what was described already in Example 2.1.2. As shown inFIGS. 14A-14Q, bivalent FAP targeted Ox40 construct showed similarbinding characteristics to Ox40 positive and negative cells asrespective clones in a conventional IgG antibody format, whereasmonovalent antibodies had a clearly reduced capacity to bind to Ox40positive cells due to the loss of avidity.

4.3.2.2 Binding to Human FAP-expressing Tumor Cells

The binding to cell surface FAP was tested using human fibroblastactivating protein (huFAP) expressing cells NIH/3T3-huFAP clone 39 orWM266-4 cells (ATCC CRL-1676). NIH/3T3-huFAP clone 39 was generated bythe transfection of the mouse embryonic fibroblast NIH/3T3 cell line(ATCC CRL-1658) with the expression vector pETR4921 to express huFAPunder 1.5 μg/mL Puromycin selection. In some assays WM266-4 cells werepre-labeled with PKH-26 Red Fluorescence Cell linker Kit (Sigma,Cat.-No. PKH26GL) as described in Example 2.3.2 to allow separation ofthese tumor cells from other cells present (e.g. human PBMC).

0.5×10⁵ NIH/3T3-huFAP clone 39 or WM266-4 cells were then added to eachwell of a round-bottom suspension cell 96-well plates (greiner bio-one,cellstar, Cat. No. 650185) and the binding assay was performed in asimilar manner as described in Example 2.3.2. Plates were centrifuged 4minutes, 400×g at 4° C. and supernatants were flicked off. Cells werewashed once with 200 μL DPBS and pellets were resuspended by a short andgentle vortex. All samples were resuspended in 50 μL/well of 4° C. coldFACS buffer containing the bispecific antigen binding molecules (primaryantibody) at the indicated range of concentrations (titrated) andincubated for 120 minutes at 4° C. Afterwards the cells were washed fourtimes with 200 μL 4° C. FACS buffer and resuspended by a short vortex.Cells were further stained with 25 μL/well of 4° C. cold secondaryantibody solution containing Fluorescein isothiocyanate(FITC)-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatIgG F(ab′)₂ fragment (Jackson ImmunoResearch, Cat. No. 109-096-098) andincubated for 30 minutes at 4° C. in the dark. Plates were finallyresuspended in 80 μL/well FACS-buffer containing 0.2 μg/mL DAPI (SantaCruz Biotec, Cat. No. Sc-3598) and acquired the same day using 5-laserLSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 15A and 15B, the FAP-targeted mono- and bivalentanti-Ox40 antigen binding molecules but not the same clones in thehuIgG1 P329GLALA format efficiently bound to human FAP-expressing targetcells. Therefore only FAP-targeted mono- and bivalent anti-Ox40 antigenbinding molecules show direct tumor-targeting properties. The bivalentconstruct (filled square) showed stronger binding to FAP than themonovalent constructs explained by a gain of avidity in the bivalentrelative to the monovalent format. This was more prominent in the highFAP expressing NIH/3T3-huFAP clone 39 cells (FIG. 15A) than in the lowerFAP expressing WM266-4 cells (FIG. 15B). A lower density of surface FAPon WM266-4 cells might not provide the close proximity of FAP moleculesto always allow bivalent binding of the anti-OX40 constructs. EC₅₀values of binding to activated human CD4 T cells and FAP positive tumorcells are summarized in Table 29.

TABLE 29 EC50 values for binding of selected aOx40 binder (clone 8H9,1G4) in a FAP targeted mono or bivalent format to cell surface human FAPand human Ox40 FAP⁺ cell OX40⁺ cell Clone Format EC₅₀ [nM] EC₅₀ [nM] 8H9hu IgG1 n.a. 0.59 (WM266-4) FAP 1 + 1 5.99 8.20 FAP 2 + 2 2.88 0.93 1G4hu IgG1 n.a. n.a. (NIH/3T3 huFAP FAP 1 + 1 3.55 49.07  clone 39) FAP 2 +2 0.77 9.374.4 Generation of Bispecific Antigen Binding Molecules Targeting OX40and Fibroblast Activation Protein (FAP) that are Bivalent for OX40 andMonovalent for FAP (2+1 Format)

Bispecific agonistic Ox40 antibodies with bivalent binding for Ox40 andmonovalent binding for FAP were prepared by applying the knob-into-holetechnology to allow the assembling of two different heavy chains.

In this example, the first heavy chain (HC 1) was comprised of one Fabunit (VHCH1) of the anti-OX40 binder 49B4 followed by Fc knob chainfused by a (G₄S) linker to a VH domain of the anti-FAP binder 28H1 or4B9. The second heavy chain (HC 2) of the construct was comprised of oneFab units (VHCH1) of the anti-OX40 binder 49B4 followed Fc hole chainfused by a (G₄S) linker to a VL domain of the anti-FAP binder 28H1 or4B9.

The generation and preparation of the FAP binders is described in WO2012/020006 A2, which is incorporated herein by reference.

The Pro329Gly, Leu234Ala and Leu235Ala mutations were introduced in theconstant region of the heavy chains to abrogate binding to Fc gammareceptors according to the method described in International PatentAppl. Publ. No. WO 2012/130831 A1. The heavy chain fusion proteins wereco-expressed with the light chain of the anti-OX40 binder 49B4 (CLVL).The resulting bispecific, construct bivalent for binding to OX40 isdepicted in FIG. 12D and the nucleotide and amino acid sequences can befound in Table 30.

In addition, an “untargeted” 2+1 construct was prepared, wherein the VHand VL domain of the anti-FAP binder were replaced by a germlinecontrol, termed DP47, not binding to the antigen.

TABLE 30 cDNA and amino acid sequences of mature bispecific bivalentanti-Ox40/monovalent anti-FAP huIgG1 P329GLALA kih antibodies (2 + 1format) and untargeted (DP47) 2 + 1 construct SEQ ID NO: DescriptionSequence 184 (49B4) VLCL-light see Table 13 chain (nucleotide sequence)304 (49B4) VHCH1 Fc CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VH (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence ofGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 1)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGG GGACAGGGCACCCTGGTCACCGTGTCCAGC305 (49B4) VHCH1 Fc hole CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VL (4B9)(nucleotide AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC sequence of heavyGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC chain 2)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG 186 (49B4) VLCL-light see Table 13chain 306 (49B4) VHCH1 Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knobVH (4B9) APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 1)YMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 307 (49B4) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VL (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 184(49B4) VLCL-light see Table 13 chain (nucleotide sequence) 308 (49B4)VHCH1 Fc CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA knob VH (28H1)AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAA (nucleotide sequence,GGCCTCCGGAGGCACATTCAGCAGCTACGCTATA heavy chain 1)AGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCG AGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCA CCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACC GCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTG ACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGT GGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGA ACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCG TGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCG GAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTGG GTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTAC GCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATG AACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTA CTGGGGCCAGGGCACCCTGGTCACCGTGTCCAGC309 (49B4) VHCH1 Fc hole CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA VL (28H1)(nucleotide AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAA sequence, heavy chainGGCCTCCGGAGGCACATTCAGCAGCTACGCTATA 2) AGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACA GCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTA CATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGAATACTACCGTGG TCCGTACGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGC CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAG TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCC CGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTG GAGGCGGCGGAAGCGGAGGAGGAGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGATCGT GCTGACCCAGTCTCCCGGCACCCTGAGCCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAG CCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGA TCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGAC TTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTG ATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAG 186 (49B4) VLCL-light see Table 13 chain 310 (49B4) VHCH1 FcQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW knob VH (28H1)VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA (heavy chain 1)DKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGG GSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSS 311 (49B4) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV VL (28H1)RQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADK (heavy chain 2)STSTAYMELSSLRSEDTAVYYCAREYYRGPYDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVI PPTFGQGTKVEIK 184 (49B4)VLCL-light see Table 13 chain (nucleotide sequence) 312 (49B4) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA knob VH (DP47)AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAA (nucleotide sequence,GGCCTCCGGAGGCACATTCAGCAGCTACGCTATA heavy chain 1)AGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCG AGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCA CCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACC GCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTG ACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGT GGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGA ACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCG TGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCG GAGGAGGGGGAAGTGGCGGCGGAGGATCTGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGTTATGCCATGAGCTGG GTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATAC TACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAG ATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTG GGGCCAAGGAACCCTGGTCACCGTCTCGAGC 313(49B4) VHCH1 Fc hole CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA VL (DP47)AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAA (nucleotide sequence,GGCCTCCGGAGGCACATTCAGCAGCTACGCTATA heavy chain 2)AGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCG AGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCA CCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACC GCCGTGTATTACTGTGCGAGAGAATACTACCGTGGTCCGTACGACTACTGGGGCCAAGGGACCACCGTG ACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTG GGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCT CGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGAG GCGGCGGAAGCGGAGGGGGAGGCTCTGAAATTGTGCTGACCCAGAGCCCCGGCACCCTGTCACTGTCTC CAGGCGAAAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGTCCAGCTCTTACCTGGCCTGGTATC AGCAGAAGCCCGGACAGGCCCCCAGACTGCTGATCTACGGCGCCTCTTCTAGAGCCACCGGCATCCCCG ATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGAC TTTGCCGTGTATTACTGCCAGCAGTACGGCAGCAGCCCCCTGACCTTTGGCCAGGGCACCAAGGTGGAA ATCAAA 186 (49B4) VLCL-light seeTable 13 chain 314 (49B4) VHCH1 Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWknob VH (DP47) VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA (heavy chain 1)DKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGG GSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS 315 (49B4) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW VL (DP47)VRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA (heavy chain 2)DKSTSTAYMELSSLRSEDTAVYYCAREYYRGPYDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

The bispecific anti-Ox40/anti-FAP 2+1 constructs were produced byco-transfecting HEK293-EBNA cells with the mammalian expression vectorsusing polyethylenimine (PEI; Polysciences Inc.). The cells weretransfected with the corresponding expression vectors in a 1:1:2 ratio(“vector HC1”:“vector HC2”:“vector LC”).

For a 200 mL production in 500 mL shake flasks, 250 million HEK293 EBNAcells were seeded 24 hours before transfection in Excell media (Sigma)with supplements. For transfection, the cells were centrifuged for 5minutes at 210×g, and supernatant was replaced by pre-warmed CD-CHOmedium (Gibco). Expression vectors were mixed in 20 mL CD-CHO medium toa final amount of 200 μg DNA. After addition of 540 μL PEI (1 mg/mL)(Polysciences Inc.), the solution was vortexed for 15 seconds andincubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂ atmosphereand shaking at 165 rpm. After the incubation, 160 mL Excell medium withsupplements (1 mM valproic acid, 5 g/1 Pep Soy, 6 mM L-Glutamine) wasadded and cells were cultured for 24 hours. 24 h after transfection thecells were supplemented with an amino acid and glucose feed at 12% finalvolume (24 mL) and 3 g/L glucose (1.2 mL from 500 g/L stock). Afterculturing for 7 days, the cell supernatant was collected bycentrifugation for 45 minutes at 2000-3000×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of the bispecific constructs from cell culture supernatantswas carried out by affinity chromatography using MabSelectSure. Theprotein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM histidine,140 mM NaCl, 0.01% Tween-20 solution of pH 6.0.

For affinity chromatography, the supernatant was loaded on a ProtAMabSelect Sure column (CV=5 mL, GE Healthcare) equilibrated with 30 mL20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5. Unbound proteinwas removed by washing with 6-10 column volumes of a buffer containing20 mM sodium phosphate, 20 mM sodium citrate (pH 7.5). The bound proteinwas eluted using either a step or a linear pH-gradient of 20 CVs (from 0to 100%) of 20 mM Sodium Citrate, 100 mM Sodium Chloride, 100 mMGlycine, 0.01% (v/v) Tween-20, pH 3.0. The column was then washed with10 column volumes of a solution containing 20 mM Sodium Citrate, 100 mMsodium chloride, 100 mM glycine, 0.01% (v/v) Tween-20, pH 3.0 followedby a re-equilibration step.

The pH of the collected fractions was adjusted by adding 1/10 (v/v) of0.5 M sodium phosphate, pH8.0. The protein was concentrated and filteredprior to loading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01%Tween20.

The protein concentration of purified bispecific constructs wasdetermined by measuring the OD at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of the bispecific constructs were analyzed byCE-SDS in the presence and absence of a reducing agent (Invitrogen)using a LabChipGXII (Caliper). The aggregate content of bispecificconstructs was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in a 25 mM potassiumphosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrochloride,0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C. (Table 31).

TABLE 31 Biochemical analysis of exemplary bispecific, tetravalentanti-Ox40/anti- FAP IgG1 P329G LALA antigen binding molecules (2 + 1constructs) Yield Monomer Clone [mg/l] [%] CE-SDS (non red) CE-SDS (red)OX40(49B4)/FAP(28H1) 8.3 97.25 100 73.7% (81.6 kDa) P329GLALA IgG1 2 + 1(0.75 HMW) 0.13% (30.9 kDa) 26.8% (29.5 kDa) OX40(49B4)/FAP(4B9) 7.8599.25 100 58.71% (80.44 kDa) P329GLALA IgG1 2 + 1 (0.75 HMW) 41.29%(28.91 kDa) OX40(49B4)/DP47 5.76 97.63 100 73.47% (81.39 kDa) P329GLALAIgG1 2 + 1 (1.33 HMW) 0.11% (30.67 kDa) 26.42% (29.42 kDa)4.5 Characterization of Bispecific 2+1 Constructs Targeting Ox40 and FAP4.5.1 Binding to FAP (Surface Plasmon Resonance)

The capacity of the bispecific constructs to bind human, murine andcynomolgus FAP was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 (Biacore) at 25° C. withHBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% Surfactant P20, (Biacore).

His-tagged human, murine or cynomolgus monkey dimeric FAP was capturedon a CM5 chip (GE Healthcare) immobilized with anti-His antibody (QiagenCat. No. 34660) by injection of 500 nM huFAP for 60 s at a flow rate of10 uL/min, 10 nM murine FAP for 20 s at a flow rate of 20 uL/min and 10nM cynoFAP for 20 s at a flow rate of 20 uL/min. Immobilization levelsfor the anti-His antibody of up to 18000 resonance units (RU) were used.The setup of the assay is shown in FIG. 12E.

Following the capture step, the bispecific constructs as well as controlmolecules were immediately passed over the chip surface at aconcentration ranging from 0.78-100 nM with a flow rate of 30 μL/minutefor 280 s and a dissociation phase of 180 s. Bulk refractive indexdifferences were corrected for by subtracting the response obtained in areference flow cell, where no FAP was immobilized. Affinity wasdetermined using the Langmuir 1:1 curve fitting. For bivalent bindingthe same 1:1 fitting was used leading to an apparent KD value.

TABLE 32 Binding of exemplary bispecific anti-Ox40/anti- FAP antigenbinding molecules to recombinant human FAP, murine FAP and cynomolgusFAP hu FAP mu FAP cyno FAP Construct K_(D) (M) K_(D) (M) K_(D) (M)OX40(49B4)/FAP(28H1) 1.9E−08 3.3E−10 3.1E−08 P329GLALA IgG1 2 + 1OX40(49B4)/FAP(4B9) 1.0E−09 1.1E−07 8.5E−10 P329GLALA IgG1 2 + 1OX40(49B4)/DP47 n.d. n.d. n.d. P329GLALA IgG1 2 + 1 Note: All K_(D)s aredependent from the specific experimental conditions.4.5.2 Binding to humanOX40—Competition Binding of Bivalent Ox40 Binding

To confirm the ability of all two anti-OX40 Fab domains to bind tohuOX40 comparable to an IgG, a cell-based FRET assay (TagLite) wasapplied. Therefore, 10000 Hek293 EBNA cells/well transfected withhuOX40-SNAP fusion and labeled with the FRET donor Terbium (Cisbio) weremixed with 0.2 nM (49B4) IgG labeled with the FRET acceptor d2 (Cisbio).Additionally, a concentration dilution ranging from 0.004-750 nM fromeither (49B4) IgG or bispecific construct 49B4/28H1 (2+1) was added andincubated for 2-4 hours at RT. The fluorescent signal was measured at620 nm for the fluorescent donor (Terbium) and at 665 nm for thefluorescent acceptor dye (M100 Pro, Tecan). The ratio of 665/620*1000was calculated, and the reference (cells only) was subtracted (FIG.13M). For EC₅₀ determination the results were analysed in Graph PadPrism5. The observed EC₅₀ values are shown in Table 33. All 2+1constructs showed a similar EC₅₀ than the bivalent IgG under theseexperimental conditions.

TABLE 33 EC₅₀ values for competition binding of IgG vs bivalent 2 + 1OX40 antigen binding molecules; t = 2 h Construct EC₅₀ (nM) 49B4 IgG10.87 (0.64-1.2) OX40(49B4)/FAP(28H1) 1.96 (1.22-3.14) P329GLALA IgG1 2 +1 OX40(49B4)/FAP(4B9) 0.93 (0.7-1.22) P329GLALA IgG1 2 + 1OX40(49B4)/FAP(DP47) 1.28 (0.95-1.72) P329GLALA IgG1 2 + 14.5.3 Simultaneous Binding to OX40 and FAP

The capacity of binding simultaneously human OX40 Fc (kih) and human FAPwas assessed by surface plasmon resonance (SPR). All SPR experimentswere performed on a Biacore T200 (Biacore) at 25° C. with HBS-EP asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%Surfactant P20 (Biacore).

Biotinylated human OX40 Fc (kih) was directly coupled to a flow cell ofa streptavidin (SA) sensor chip. Immobilization levels up to 1000resonance units (RU) were used.

The bispecific antibodies targeting OX40 and FAP were passed over thechip surface at a concentration of 250 nM with a flow rate of 30μL/minute for 90 seconds and dissociation was set to zero sec. Human FAPwas injected as second analyte with a flow rate of 30 μL/minute for 90seconds at a concentration of 250 nM (see FIG. 12F). The dissociationwas monitored for 120 sec. Bulk refractive index differences werecorrected for by subtracting the response obtained in a reference flowcell, where no protein was immobilized.

All bispecific constructs could bind simultaneously to human OX40 andhuman FAP (FIGS. 13J-13L).

4.5.4 Binding on Cells

4.5.4.1 Binding to Naïve Versus Activated Human PBMCs of BispecificAntibodies Targeting OX40 and FAP

Human PBMC were isolated by ficoll density gradient centrifugation asdescribed in Example 2.1.2. PBMCs were used directly after isolation(binding on resting human PBMCs) or they were stimulated to receive astrong human Ox40 expression on the cell surface of T cells (binding onactivated human PBMCs). Therefore naïve PBMCs were cultured for fourdays in T cell medium supplied with 200 U/mL Proleukin and 2 μg/mL PHA-Lin 6-well tissue culture plate and then 1 day on pre-coated 6-welltissue culture plates [4 μg/mL anti-human CD3 (clone OKT3) and 2 μg/mLanti-human CD28 (clone CD28.2)] in T cell medium supplied with 200 U/mLProleukin at 37° C. and 5% CO₂.

For detection of OX40 naïve human PBMC and activated human PBMC weremixed. To enable distinction of naïve from activated human PBMC naïvecells were labeled prior to the binding assay using the eFluor670 cellproliferation dye (eBioscience, Cat.-No. 65-0840-85). A 1 to 1 mixtureof 1×10⁵ naïve, eFluor670 labeled human PBMC and unlabeled activatedhuman PBMC were then added to each well of a round-bottom suspensioncell 96-well plates (greiner bio-one, cellstar, Cat. No. 650185) and thebinding assay was performed as described in Example 2.1.2. A 1 to 1mixture of 1×105 naïve, eFluor670 labeled human PBMC and unlabeledactivated human PBMC were then added to each well of a round-bottomsuspension cell 96-well plates (greiner bio-one, cellstar, Cat. No.650185) and binding assay was performed as described in section 2.1.2.

Primary antibodies were titrated anti-Ox40 antibody constructs,incubated for 120 minutes at 4° C. Secondary antibody solution was amixture of fluorescently labeled anti-human CD4 (clone RPA-T4, mouseIgG1 k, BioLegend, Cat.-No. 300532), anti-human CD8 (clone RPa-T8, mouseIgG1k, BioLegend, Cat.-No. 3010441) and Fluorescein isothiocyanate(FITC)-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatIgG F(ab′)₂ fragment (Jackson ImmunoResearch, Cat.-No. 109-096-098),incubated for 60 minutes at 4° C. in the dark. Plates were finallyresuspended in 90 μL/well FACS-buffer containing 0.2 μg/mL DAPI (SantaCruz Biotec, Cat. No. Sc-3598) and acquired the same day using 5-laserLSR-Fortessa (BD Bioscience with DIVA software).

As shown in FIGS. 14J and 14Q, no antigen binding molecule specific forOx40 bound to resting human CD4⁺ T-cells or CD8⁺ T-cells. In contrast,all antigen binding molecules bound to activated CD8⁺ or CD4⁺ T-cells.Binding to CD4⁺ T-cells was much stronger than that to CD8⁺ T cellssimilar to what was described already in Example 4.3.2.1. As shown inFIGS. 14K and 14M, the bivalent FAP-targeted OX40 constructs showedstronger binding characteristics to OX40 positive cells as respectiveclone in a monovalent antibody format, due to the gain of avidity. Allformats of a 2+1 design bound with similar strength to OX40 positivecells, independently of the binding moiety of the second specificity(FIGS. 14O and 14Q).

4.5.4.2 Binding to Human FAP-expressing Tumor Cells

The binding to cell surface FAP was tested using human fibroblastactivating protein (huFAP) expressing WM266-4 cells (ATCC CRL-1676). Thelack of binding to OX40 negative FAP negative tumor cells was testedusing A549 NucLight™ Red Cells (Essenbioscience, Cat. No. 4491)expressing the NucLight Red fluorescent protein restricted to thenucleus to allow separation from unlabeled human FAP positive WM266-4cells. Parental A549 (ATCC CCL-185) were transduced with the EssenCellPlayer NucLight Red Lentivirus (Essenbioscience, Cat. No. 4476;EF1α, puromycin) at an MOI of 3 (TU/cell) in the presence of 8 μg/mlpolybrene following the standard Essen protocol. This resulted in ≥70%transduction efficiency.

A mixture of 5×104 unlabeled WM266-4 cells and unlabeled A549 NucLight™Red Cells in FACS buffer were added to each well of a round-bottomsuspension cell 96-well plates (Greiner bio-one, Cellstar, Cat. No.650185) and the binding assay was performed in a similar manner asdescribed in Example 4.3.2.2. Plates were centrifuged 4 minutes, 400×gat 4° C. and supernatants were flicked off. Cells were washed once with200 μL DPBS and pellets were resuspended by a short and gentle vortex.All samples were resuspended in 50 μL/well of 4° C. cold FACS buffercontaining the bispecific antigen binding molecules (primary antibody)at the indicated range of concentrations (titrated) and incubated for120 minutes at 4° C. Afterwards the cells were washed four times with200 μL/well 4° C. FACS buffer and resuspended by a short vortex. Cellswere further stained with 25 μL/well of 4° C. cold secondary antibodysolution containing Fluorescein isothiocyanate (FITC)-conjugatedAffiniPure anti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂fragment (Jackson ImmunoResearch, Cat. No. 109-096-098) and incubatedfor 60 minutes at 4° C. in the dark. Plates were finally resuspended in90 μL/well FACS-buffer containing 0.2 μg/mL DAPI (Santa Cruz Biotec,Cat. No. Sc-3598) and acquired the same day using 5-laser LSR-Fortessa(BD Bioscience with DIVA software).

As shown in FIGS. 15C and 15E, the FAP-targeted mono- and bivalentanti-OX40 antigen binding molecules bounds efficiently to humanFAP-expressing target cells. Therefore only FAP-targeted mono- andbivalent anti-OX40 antigen binding molecules show direct tumor-targetingproperties. The high affinity FAP binding clone 4B9 showed in amonovalent construct a stronger binding to human FAP than the respectiveFAP binding clone 28H1 in the same monovalent format (FIG. 15E). No FAPwas detected for 2+1 constructs lacking a FAP binding domain (opencircle, FIG. 15F). EC₅₀ values of binding to activated human CD4⁺ Tcells and FAP positive tumor cells are summarized in Table 34.

TABLE 34 EC₅₀ values for binding of aOx40 binder (clone 49B4) in a FAPtargeted mono or bivalent format to cell surface human FAP and humanOx40 FAP⁺ cell OX40⁺ cell Clone Format EC₅₀ [nM] EC₅₀ [nM] 49B4, 28H1FAP 1 + 1 1.0 123.1 FAP 2 + 1 70.8 6.5 FAP 2 + 2 1.2 6.1 49B4, 4B9 FAP2 + 1 4.1 9.0 49B4, DP47 FAP 2 + 1 5.99 9.0

Example 5 Functional Properties of Bispecific Anti-human OX40 BindingMolecules

5.1 HeLa Cells Expressing Human OX40 and Reporter Gene NFκB-luciferase

As shown in Example 3.1, a limited, dose dependent NFκB activation wasinduced by addition of anti-OX40 P329GLALA huIgG1 antibodies to theHeLa_hOX40_NFkB_Luc1 reporter cell line. Hyper-crosslinking of anti-OX40antibodies by anti-human IgG specific secondary antibodies stronglyincreased the induction of NFκB-mediated luciferase-activation in aconcentration-dependent manner. Consequently, we tested the NFκBactivating capacity of selected anti-OX40 binders (8H9, 1G4, 49B9) in amonovalent and bivalent FAP-targeted format alone and withhyper-crosslinking of the constructs by either a secondary antibody or aFAP⁺ tumor cell line.

As described in Example 3.1, adherent HeLa_hOX40_NFκB_Luc1 cells werecultured over night at a cell density of 0.3*10⁵ cells per well and werestimulated for 5 to 6 hours with assay medium containing titratedanti-OX40 binders (clone 8H9 and 1G4) in the FAP targeted monovalent(FAP 1+1) and bivalent (FAP 2+2) format and as P329GLALA hu IgG1constructs. For testing the effect of hyper-crosslinking by secondaryantibodies, 25 μL/well of medium containing secondary antibodyanti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂ fragment (JacksonImmunoResearch, 109-006-098) was added in a 1:2 ratio (primary tosecondary antibodies). To test the effect of hyper-crosslinking by cellsurface FAP binding, 25 μL/well of medium containing FAP⁺ tumor cells(WM266-4 and/or NIH/3T3-huFAP clone 39) were co-cultured in a 2 to 1ratio (twice as much FAP⁺ tumor cells than reporter cells per well).Activated NFκB was quantified by measuring light emission usingluciferase 1000 assay system and the reporter lysis buffer (bothPromega, Cat.-No. E4550 and Cat-No: E3971) as described in Example 3.1.

As shown in FIGS. 16A-16G, the presence of all anti-OX40 constructsinduced a limited NFkB activation. Hyper-crosslinking via secondaryanti-huIgG Fcγ-specific antibody increased this NFkB activation for bothbinders in a huIgG1 P329GLALA as well as in a FAP targeted mono/bivalentformat. Monovalent binding to OX40 was thereby less efficient thanbivalent binding to OX40, which showed the necessity of OX40 receptoroligomerization to fully activate the OX40 signaling axis.FAP-expressing tumor cell strongly increased induction of NFκB-mediatedluciferase-activation in a concentration-dependent manner when FAPtargeted molecules (filled square and triangle) were used. No sucheffect was seen when the same clones in a non-targeted huIgG1 P329GLALAformat were used as the construct could not be further hyper-crosslinkedby FAP⁺ tumor cells. Again the bivalent molecule was superior to themonovalent molecule. A high expression of FAP ensured highercross-linking and thus a better agonistic effect of the FAP targetedconstruct (compare filled diamond for FAP high NIH/3T3-huFAP clone 39and FAP positive WM266-4 cells). The bivalent FAP targeted constructshowed in the presence of FAP⁺ tumor cells a peak activity at aconcentration of ˜0.1 to 1 nM. Further increase of compoundconcentration actually decreased its ability to induce NFκB. Mostlikely, bivalent constructs binding to only one target (FAP or OX40)were present at higher concentrations, out-competing constructs bindingsimultaneously to FAP and OX40. This loss of cross-linking reduced inturn the agonistic OX40 signaling.

In a further experiment, adherent HeLa_hOX40_NFκB_Luc1 cells werecultured over night at a cell density of 0.2*10⁵ cells per well and werestimulated for 5 hours with assay medium containing titrated anti-OX40binders (clone 49B9) in the FAP targeted monovalent (FAP 1+1) andbivalent (FAP 2+1 and FAP 2+2) format and as P329GLALA hu IgG1constructs. FAP was targeted in the 2+1 with two different FAP bindingclones, 28H1 with low affinity to FAP and 4B9 with high affinity forFAP. For testing the effect of hyper-crosslinking by secondaryantibodies, 25 μL/well of medium containing secondary antibodyanti-human IgG Fcγ-fragment-specific goat IgG F(ab′)₂ fragment (JacksonImmunoResearch, 109-006-098) was added in a 1:2 ratio (primary tosecondary antibodies). To test the effect of hyper-crosslinking by cellsurface FAP binding, 25 μL/well of medium containing FAP⁺ tumor cells(NIH/3T3-huFAP clone 19) were co-cultured in a 4 to 1 ratio (four timeas much FAP⁺ tumor cells than reporter cells per well). Activated NFκBwas quantified by measuring light emission using luciferase 1000 assaysystem and the reporter lysis buffer (both Promega, Cat.-No. E4550 andCat-No: E3971) as described in Example 3.1.

As shown in FIGS. 16H-16N, also in this experiment the presence of allanti-OX40 constructs induced a limited NFkB activation.Hyper-crosslinking via secondary anti-huIgG Fcγ-specific antibodyincreased this NFkB activation for all binders in a FAP targetedmono/bivalent format. Monovalent binding to OX40 was thereby lessefficient than bivalent binding to OX40, which showed the necessity ofOX40 receptor oligomerization to fully activate the OX40 signaling axis(FIG. 16J, compare EC₅₀ values). FAP-expressing tumor cells stronglyincreased induction of NFκB-mediated luciferase-activation in aconcentration-dependent manner when FAP targeted molecules (filledsquare, triangle, semi-filled circles) were used. No such effect wasseen when in the 2+1 format the FAP binding moiety was replaced by anon-binding DP47 unit (open circle) as the construct could not befurther hyper-crosslinked by FAP⁺ tumor cells. Again the bivalentmolecule was superior to the monovalent molecule (compare EC₅₀ values).The monovalent FAP targeted and bivalent OX40 targeted construct (2+1)showed in the presence of FAP⁺ tumor cells the highest plateau activity.In contrast to the monovalent OX40 binding 1+1 construct the bivalentbinding to OX40 of the 2+1 format increased the agonistic capacity byoligomerization of OX40 (effect on EC₅₀ value). Due to the monovalentbinding of the 2+1 constructs to FAP, however, it seems that twice asmuch molecules could be crosslinked compared to the bivalent FAP binding2+2 format which could explain the higher plateau OX40 activation (FIG.16K, higher plateau).

5.2 OX40 Mediated Costimulation of Suboptimally TCR TriggeredPre-activated Human CD4 T Cells

Selected binders (clone 8H9 and 1G4) in a FAP targeted monovalent orbivalent format were also tested for their ability to co-activate Tcells when they were surface immobilized.

As described in Example 3.2, pre-activated CFSE-labeled OX40 positiveCD4 T-cells were stimulated for 72 hours with a suboptimal concentrationof plate-immobilized anti-CD3 antibodies in the presence of titratedanti-Ox40 antibodies immobilized on the plate surface. Effects on T-cellsurvival and proliferation were analyzed through monitoring of totalcell counts and CFSE dilution in living cells by flow cytometry.Additionally, cells were co-stained with fluorescently-labeledantibodies against T-cell activation and differentiation markers, e.g.CD127, CD45RA, Tim-3, CD62L and OX40 itself.

Co-stimulation with plate-immobilized bispecific anti-OX40 antigenbinding molecules strongly enhanced suboptimal stimulation ofpre-activated human CD4 T cells with plate-immobilized anti-human CD3 ina dose dependent manner (FIGS. 17A and 17B). T-cells proliferatedstronger, showed a more mature phenotype with a higher percentage ofCD127 low T cells, Tim-3 positive and OX40 positive activated cells (forclone 8H9 only shown for increase in granularity (SSC)). Monovalentbinding (triangle symbol) to OX40 was thereby less efficient thanbivalent binding (square symbol) to OX40, which showed the necessity ofOX40 receptor oligomerization to fully activate the Ox40 signaling axis.Half-maximal changes in all analyzed parameters of T-cell activationwere achieved at concentrations ranging from 3 to 2300 pM and aresummarized for clone 1G4 in FIGS. 18A-18D and Table 35.

TABLE 35 EC₅₀ values of rescuing suboptimal TCR stimulation withplate-immobilized FAP targeted mono and bivalent anti-Ox40 (clone 1G4)constructs Clone Format EC₅₀ [nM] +/−SEM 1G4 hu IgG1 0.37 0.03 FAP 1 + 12.23 0.05 FAP 2 + 2 0.75 0.165.3 Ox40 Mediated Costimulation of Suboptimally TCR Triggered RestingHuman PBMC and Hypercrosslinking by Cell Surface FAP

It was shown in Example 5.1 that addition of FAP⁺ tumor cells canstrongly increase the NFkB activity induced by FAP targeted mono andbivalent anti-OX40 constructs in human OX40 positive reporter cell linesby providing strong oligomerization of OX40 receptors. Likewise, wetested FAP targeted mono (1+1) and bivalent (2+1, 2+2) anti-OX40constructs in the presence of NIH/3T3-huFAP clone 39 cells for theirability to rescue suboptimal TCR stimulation of resting human PBMCcells.

Human PBMC preparations contain (1) resting OX40 negative CD4⁺ and CD8⁺T cells and (2) antigen presenting cells with various Fc-γ receptormolecules on their cell surface e.g. B cells and monocytes. Anti-humanCD3 antibody of human IgG1 isotype can bind with its Fc part to thepresent Fc-γ receptor molecules and mediate a prolonged TCR activationon resting Ox40 negative CD4⁺ and CD8⁺ T cells. These cells then startto express OX40 within several hours. Functional agonistic compoundsagainst OX40 can signal via the OX40 receptor present on activated CD8⁺and CD4⁺ T cells and support TCR-mediated stimulation.

Resting CFSE-labeled human PBMC were stimulated for four to five dayswith a suboptimal concentration of anti-CD3 antibody in the presence ofirradiated FAP⁺ NIH/3T3-huFAP clone 39 cells and titrated anti-Ox40constructs. Effects on T-cell survival and proliferation were analyzedthrough monitoring of total cell counts and CFSE dilution in livingcells by flow cytometry. Additionally, cells were co-stained withfluorescently-labeled antibodies against T-cell activation andmaturation marker CD25, Granzyme B, CD62L and CD127. In a secondexperiment, cells were co-stained with fluorescently-labeled antibodiesagainst T-cell activation and maturation marker CD25, and Tim-3.

Mouse embryonic fibroblast NIH/3T3-huFAP clone 39 cells (Example4.3.2.2) were harvested using cell dissociation buffer (Invitrogen,Cat.-No. 13151-014) for 10 minutes at 37° C. Cells were washed once withDPBS. NIH/3T3-huFAP clone 39 cells were cultured at a density of 0.2*10⁵cells per well in T cell media in a sterile 96-well round bottomadhesion tissue culture plate (TPP, Cat. No. 92097) over night at 37° C.and 5% CO₂ in an incubator (Hera Cell 150). The next day they wereirradiated in an xRay irradiator using a dose of 4500 RAD to preventlater overgrowth of human PBMC by the tumor cell line.

Human PBMCs were isolated by ficoll density centrifugation and werelabeled with CFSE as described in Example 2.1.2. Cells were added toeach well at a density of 0.75*10⁵ cells per well (first experiment) or0.5*10⁵ cells per well (second experiment). Anti-human CD3 antibody(clone V9, human IgG1) at a final concentration of [10 nM] and FAPtargeted mono- and bivalent anti-OX40 antigen binding molecules wereadded at the indicated concentrations. Cells were activated for four tofive days at 37° C. and 5% CO₂ in an incubator (Hera Cell 150). Then,cells were surface-stained with fluorescent dye-conjugated antibodiesanti-human CD4 (clone RPA-T4, BioLegend, Cat.-No. 300532), CD8 (cloneRPa-T8, BioLegend, Cat.-No. 3010441), CD25 (clone M-A251, BioLegend,Cat.-No. 356112), CD127 (clone A019D5, Biolegend, Cat.No. 351234) andCD62L (clone DREG 56, Biolegend, Ca.No. 304834) or Tim-3 (clone F38-E2E,Biolegend, Cat.No. 345012) for 30 min at 4° C. For permeabilizing thecell membrane, cell pellets were washed twice with FACS buffer, thenresuspended in 50 μL/well freshly prepared FoxP3 Fix/Perm buffer(eBioscience, Cat.-No. 00-5123 and 00-5223) for 45 min at roomtemperature in the dark. After three times washing with Perm-Wash buffer(eBioscience, Cat.-No. 00-8333-56), cells were stained intracellularwith 25 μL/well Perm-Wash Buffer containing anti-human Granzyme Bantibody (clone GB-11, BD Bioscience, Cat. No. 561142) for 1 h at roomtemperature in the dark. Cells were resuspended in 85 μL/well FACSbuffer and acquired using a 5-laser Fortessa flow cytometer (BDBioscience with DIVA software).

As shown in FIGS. 20A-20H, costimulation with non-targeted anti-Ox40(8H9) huIgG1 P329GLALA (open square) did not rescue suboptimally TCRstimulated CD4 and CD8 T cells. Hyper-crosslinking of the FAP targetedmono (filled triangle) and bivalent (filled square) anti-OX40 constructsby the present NIH/3T3-huFAP clone 39 cells strongly promotedproliferation, survival and induced an enhanced activated phenotype inhuman CD4 and CD8 T cells. For high affinity clone 8H9 (FIGS. 20A-20H),monovalent and bivalent bispecific antigen binding molecule had acomparable ability to rescue suboptimal TCR stimulation. Both constructsshowed again a peak activity at ˜0.1-1 nM and a reduced response athigher concentration. Similar to the findings in the NFκB reporter cellline (FIGS. 16A-16M) this might be a consequence of competition fortarget binding between constructs that bind only to FAP or OX40 andthose that bind simultaneously to both targets and provide thus thenecessary cross-linking. This effect was less prominent when the lowaffinity clone 1G4 was tested in a FAP targeted monovalent versus abivalent anti-OX40 construct (FIGS. 21A-21C). Here, the monovalentantibody was clearly inferior to the bivalent construct. This can bebest appreciated when the agonistic capacity of each construct wasquantified as area under the curve and plotted against each other (FIG.21A-21C).

The data as obtained in the second experiment are shown in FIGS. 21D-21Hand FIGS. 21J-21N, respectively. As shown in FIGS. 21D-21H,costimulation with non-targeted anti-Ox40 (49B4) 2+1 DP47 format (opencircle) did not rescue suboptimally TCR stimulated CD4 and CD8 T cells.Hyper-crosslinking of the FAP targeted mono (filled triangle) andbivalent (filled square, semi-filled circle) anti-OX40 constructs by thepresent NIH/3T3-huFAP clone 39 cells strongly promoted survival andinduced an enhanced activated phenotype in human CD4 and CD8 T cells.Monovalent anti-OX40 construct (1+1; filled triangle) was less able torescue TCR stimulation than bivalent anti-OX40 targeting constructs(semi-filled circle, filled square). The bivalently to FAP binding 2+2construct was already able at lower concentrations to rescue suboptimalTCR stimulation compared to the monovalently to FAP binding 2+1constructs. In the 2+1 format the high affinity FAP binding clone 4B9was clearly superior to the low affinity clone 28H1 (FIGS. 21J-21N).This suggests that the EC₅₀ values of the observed bioactivity weredriven by the binding to FAP (2+2>2+1 (4B9)>2+1 (28H1)). This can bebest appreciated when the agonistic capacity of each construct wasquantified for the analyzed markers as area under the curve and plottedagainst each other (FIG. 21P).

5.4 Prevention of ADCC by Using an Human IgG1 P329GLALA Format

Antibodies of the human IgG1 class can induce antibody dependent celldeath (ADCC) of antigen positive target cells by binding to FcγReceptors (FcγR) on ADCC competent cells, e.g. NK cells. Thus, an ADCCcompetent Ox40 antibody could mediate lysis of recently activated, Ox40⁺T cells and diminish the pool of tumor-reactive T cells. Theintroduction of the IgG1P329GLALA mutation to the Fc part of theantibody prevents binding to FcγR (International Patent Appl. Publ. No.WO 2012/130831 A1), and thus ADCC in the presence of NK cells. However,binding to the FcN receptor is not altered to ensure IgG likepharmacokinetics of the antibody.

Selected clones were converted to a conventional human IgG1 format totest for their ability to induce ADCC. To test ADCC competence, PKh26labeled OX40 positive tumor cells (HeLa_hOX40_NFkB_Luc1 reporter cellline) and freshly isolated NK cells were cocultured at an E to T ratioof 3 to 1 in the presence of a serial dilution row of anti OX40antibodies (human IgG1 or human IgG1-P329GLALA format). The release oflactate dehydrogenase (LDH) and the amount of DAPI positive tumor cellswas used to quantify NK cell mediated ADCC.

Briefly, HeLa_hOX40_NFkB_Luc1 cells (Example 3.1) were labeled using thePKH-26 Red Fluorescence Cell linker Kit (Sigma, Cat.-No. PKH26GL) asdescribed in Example 2.3.2. PKH-26 labeled HeLa_hOx40_NFkB_luc1 cellswere seeded at a density of 0.5*10⁵ cells per well in AIM V media(Gibco, Cat.No. 12055-09) in a sterile 96-well round bottom adhesiontissue culture plate (TPP, Cat. No. 92097) over night at 37° C. and 5%CO₂ in an incubator (Hera Cell 150). The next day, human PBMCs wereisolated by ficoll density gradient centrifugation as described inExample 2.1.2. NK cell isolation was performed using the MACS negativeNK cell isolation kit, human (Miltenyi Biotec, CatNo. 130-092-657)according to manufacturer instructions. NK cells were added at a densityof 1.5*10⁵ cells per well in AIM V media resulting in an E to T rationof 3 to 1. Anti-OX40 antibodies (human IgG1 or human IgG1 P329GLALA)were added at the indicated concentrations and plates were incubatedover night at 37° C. and 5% CO₂ in an incubator (Hera Cell 150). After 4hrs, 100 μL supernatant was sampled for LDH analysis and media wasreplaced with fresh AIM-V media. LDH activity was quantified using thecytotoxicity detection kit—LDH (Roche, Cat.No. 11644793001) according tomanufacturers's instructions on a SpectraMax M5/M5^(e) (MolecularDevices) microplate reader (Filter 490 nm-650 nM, 1 ms integrationtime).

After 24 hours cells were detached using Trypsin. Cells were stainedwith fluorescently labeled anti-human CD56 (clone NCAM16.2, mouse IgG1κ, BioLegend, Cat.-No. 562751), anti-human CD25 (clone MA251, mouse IgG1κ, BioLegend, Cat.-No. 356116) and anti-human CD69 (clone FN50, mouseIgG1 κ, BioLegend, Cat.-No. 356116) incubated for 20 minutes at 4° C. inthe dark. Plates were finally resuspended in 80 μL/well FACS-buffercontaining 0.2 μg/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) andacquired the same day using 5-laser LSR-Fortessa (BD Bioscience withDIVA software).

All Ox40 antibodies in an ADCC competent human IgG1 format were able toinduce lysis of Ox40 positive cells to a similar extent then an ADCCcompetent reference antibody (GA201) against EGFR. Clones in anIgGP329GLALA format did not mediate ADCC (see FIGS. 22A and 22B).

The introduction of the IgG1 P329GLALA mutation to the Fc part of ourtargeted formats prevents binding to FcγR (International Patent Appl.Publ. No. WO 2012/130831 A1), and thus ADCC in the presence of NK cells.However, binding to the FcN receptor is not altered to ensure IgG likepharmacokinetics of the antibody. In contrast to already existing OX40antibodies the hypercrosslinking that is necessary for optimal agonisticOX40 signaling was provided in the bispecific antibodies of theinvention by binding to FAP positive tumor cells or fibroblasts. FAPpositivity, either on tumor associated fibroblasts or on tumor cellsthemselves, is reported for many tumor indications. Thus, the bispecificformat has the potential for strong OX40 mediated agonism in the tumormicroenvironment in the absence of systemic activation, which mightprevent immune related toxicities. Contrary to conventional anti-OX40antibodies the bispecific antigen binding molecules of the invention donot induce ADCC of recently activated OX40 positive effector T cells.Thus, the bispecific antibodies may have the potential to reactivate apreexisting, but suppressed adaptive immune response against the tumorcells and can be effectively used for the treatment of cancer patients.

Example 6 Generation of 4-1BB Antibodies and Tool Binders

6.1 Preparation, Purification and Characterization of Antigens andScreening Tools for the Generation of Novel 4-1BB Binders by PhageDisplay

DNA sequences encoding the ectodomains of human, mouse or cynomolgus4-1BB (Table 36) were subcloned in frame with the human IgG1 heavy chainCH2 and CH3 domains on the knob (Merchant et al., 1998). An AcTEVprotease cleavage site was introduced between an antigen ectodomain andthe Fc of human IgG1. An Avi tag for directed biotinylation wasintroduced at the C-terminus of the antigen-Fc knob. Combination of theantigen-Fc knob chain containing the S354C/T366W mutations, with a Fchole chain containing the Y349C/T366S/L368A/Y407V mutations allowsgeneration of a heterodimer which includes a single copy of 4-1BBectodomain containing chain, thus creating a monomeric form of Fc-linkedantigen (FIG. 1A). Table 37 shows the cDNA and amino acid sequences ofthe antigen Fc-fusion constructs.

TABLE 36 Amino acid numbering of antigen ectodomains (ECD) and theirorigin SEQ ID NO: Construct Origin ECD 39 human 4-1BB Synthetizedaccording aa 24-186 ECD to Q07011 240 cynomolgus isolated fromcynomolgus aa 24-186 4-1BB ECD blood 241 murine 4-1BB Synthetizedaccording aa 24-187 ECD to P20334

TABLE 37 cDNA and amino acid sequences of monomeric antigen Fc(kih)fusion molecules (produced by combination of one Fc hole chain with oneantigen Fc knob chain) SEQ ID NO: Antigen Sequence 124 Nucleotide seeTable 2 sequence Fc hole chain 242 NucleotideCTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCACCTTCT sequenceGCGACAACAACCGGAACCAGATCTGCAGCCCCTGCCCCC human 4-1BBCCAACAGCTTCAGCTCTGCCGGCGGACAGCGGACCTGCG antigen FcACATCTGCAGACAGTGCAAGGGCGTGTTCAGAACCCGGA knob chainAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGACCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGGCACCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAA TGGCACGAG 243 NucleotideTTGCAGGATCTGTGTAGTAACTGCCCAGCTGGTACATTCT sequenceGTGATAATAACAGGAGTCAGATTTGCAGTCCCTGTCCTCC cynomolgus 4-AAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGTGA 1BB antigenCATATGCAGGCAGTGTAAAGGTGTTTTCAAGACCAGGAA Fc knob chainGGAGTGTTCCTCCACCAGCAATGCAGAGTGTGACTGCATTTCAGGGTATCACTGCCTGGGGGCAGAGTGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAATTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAATGACCAGAAACGTGGCATCTGTCGCCCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGCGGACCATCTCCAGCCGACCTCTCTCCAGGAGCATCCTCTGCGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGG CACGAG 244 NucleotideGTGCAGAACAGCTGCGACAACTGCCAGCCCGGCACCTTC sequenceTGCCGGAAGTACAACCCCGTGTGCAAGAGCTGCCCCCCC murine 4-1BBAGCACCTTCAGCAGCATCGGCGGCCAGCCCAACTGCAAC antigen FcATCTGCAGAGTGTGCGCCGGCTACTTCCGGTTCAAGAAGT knob chainTCTGCAGCAGCACCCACAACGCCGAGTGCGAGTGCATCGAGGGCTTCCACTGCCTGGGCCCCCAGTGCACCAGATGCGAGAAGGACTGCAGACCCGGCCAGGAACTGACCAAGCAGGGCTGTAAGACCTGCAGCCTGGGCACCTTCAACGACCAGAACGGGACCGGCGTGTGCCGGCCTTGGACCAATTGCAGCCTGGACGGGAGAAGCGTGCTGAAAACCGGCACCACCGAGAAGGACGTCGTGTGCGGCCCTCCCGTGGTGTCCTTCAGCCCTAGCACCACCATCAGCGTGACCCCTGAAGGCGGCCCTGGCGGACACTCTCTGCAGGTCCTGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGAT TGAATGGCACGAG 128 Fc hole chainsee Table 2 245 human 4-1BB LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICantigen Fc RQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDC knob chainKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDI FEAQKIEWHE 246 cynomolgus 4-LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDICR 1BB antigenQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQDCK Fc knob chainQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIF EAQKIEWHE 247 murine 4-1BBVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNICR antigen FcVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDCRP knob chainGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVLVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDI FEAQKIEWHE

All 4-1BB-Fc-fusion molecule encoding sequences were cloned into aplasmid vector, which drives expression of the insert from an MPSVpromoter and contains a synthetic polyA signal sequence located at the3′ end of the CDS. In addition, the vector contains an EBV OriP sequencefor episomal maintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a2:1:0.05 ratio (“antigen ECD-AcTEV-Fc knob”:“Fc hole”:“BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and the supernatant wasreplaced by pre-warmed CD CHO medium. Expression vectors wereresuspended in 20 mL of CD CHO medium containing 200 μg of vector DNA.After addition of 540 μL of polyethylenimine (PEI), the solution wasvortexed for 15 seconds and incubated for 10 minutes at roomtemperature. Afterwards, cells were mixed with the DNA/PEI solution,transferred to a 500 mL shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO₂ atmosphere. After the incubation, 160 mLof F17 medium was added and cells were cultured for 24 hours. Theproduction medium was supplemented with 5 μM kifunensine. One day aftertransfection, 1 mM valproic acid and 7% Feed were added to the culture.After 7 days of culturing, the cell supernatant was collected byspinning down cells for 15 min at 210 g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unboundprotein was removed by washing with at least 10 column volumes of 20 mMsodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride containingbuffer (pH 7.5). The bound protein was eluted using a linear pH-gradientof sodium chloride (from 0 to 500 mM) created over 20 column volumes of20 mM sodium citrate, 0.01% (v/v) Tween-20, pH 3.0. The column was thenwashed with 10 column volumes of 20 mM sodium citrate, 500 mM sodiumchloride, 0.01% (v/v) Tween-20, pH 3.0. The pH of collected fractionswas adjusted by adding 1/40 (v/v) of 2M Tris, pH8.0. The protein wasconcentrated and filtered prior to loading on a HiLoad Superdex 200column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodiumchloride, 0.02% (w/v) sodium azide solution of pH 7.4.

6.2 Selection of 4-1BB-Specific 12B3, 25G7, 11D5, 9B11 and 20G2Antibodies from Generic F(Ab) Libraries

The antibodies 11D5, 9B11, and 12B3 with specificity for human andcynomolgus 4-1BB were selected from a generic phage-displayed antibodylibrary (DP88-4) in the Fab format. From the same library, an additionalantibody, clone 20G2, with reactivity to murine 4-1BB was selected aswell. This library was constructed on the basis of human germline genesusing the V-domain pairing Vk1_5 (kappa light chain) and VH1_69 (heavychain) comprising randomized sequence space in CDR3 of the light chain(L3, 3 different lengths) and CDR3 of the heavy chain (H3, 3 differentlengths). Library generation was performed by assembly of 3PCR-amplified fragments applying splicing by overlapping extension (SOE)PCR. Fragment 1 comprises the 5′ end of the antibody gene includingrandomized L3, fragment 2 is a central constant fragment spanning fromL3 to H3 whereas fragment 3 comprises randomized H3 and the 3′ portionof the antibody gene. The following primer combinations were used togenerate these library fragments for DP88-4 library: fragment 1 (forwardprimer LMB3 combined with reverse primers Vk1_5_L3r_S or Vk1_5_L3r_SY orVk1_5_L3r_SPY), fragment 2 (forward primer RJH31 combined with reverseprimer RJH32) and fragment 3 (forward primers DP88-v4-4 or DP88-v4-6 orDP88-v4-8 combined with reverse primer fdseqlong), respectively. PCRparameters for production of library fragments were 5 min initialdenaturation at 94° C., 25 cycles of 1 min 94° C., 1 min 58° C., 1 min72° C. and terminal elongation for 10 min at 72° C. For assembly PCR,using equimolar ratios of the gel-purified single fragments as template,parameters were 3 min initial denaturation at 94° C. and 5 cycles of 30s 94° C., 1 min 58° C., 2 min 72° C. At this stage, outer primers (LMB3and fdseqlong) were added and additional 20 cycles were performed priorto a terminal elongation for 10 min at 72° C. After assembly ofsufficient amounts of full length randomized Fab constructs, they weredigested NcoI/NheI and ligated into similarly treated acceptor phagemidvector. Purified ligations were used for ˜60 transformations intoelectrocompetent E. coli TG1. Phagemid particles displaying the Fablibrary were rescued and purified by PEG/NaCl purification to be usedfor selections. These library construction steps were repeated threetimes to obtain a final library size of 4.4×10⁹. Percentages offunctional clones, as determined by C-terminal tag detection in dotblot, were 92.6% for the light chain and 93.7% for the heavy chain,respectively.

The antibody 25G7 with specificity for human and cynomolgus 4-1BB wasselected from a generic phage-displayed antibody library (λ-DP47) in theFab format. This library was constructed on the basis of human germlinegenes using the V-domain pairing Vl3_19 (lambda light chain) and VH3_23(heavy chain) comprising randomized sequence space in CDR3 of the lightchain (L3, 3 different lengths) and CDR3 of the heavy chain (H3, 3different lengths). Library generation was performed by assembly of 3PCR-amplified fragments applying splicing by overlapping extension (SOE)PCR. Fragment 1 comprises the 5′ end of the antibody gene includingrandomized L3, fragment 2 is a central constant fragment spanning fromL3 to H3 whereas fragment 3 comprises randomized H3 and the 3′ portionof the antibody gene. The following primer combinations were used togenerate these library fragments for λ-DP47 library: fragment 1 (forwardprimer LMB3 combined with reverse primers Vl_3_19_L3r_V orVl_3_19_L3r_HV or Vl_3_19_L3r_HLV), fragment 2 (forward primer RJH80combined with reverse primer MS63) and fragment 3 (forward primersDP47-v4-4 or DP47-v4-6 or DP47-v4-8 combined with reverse primerfdseqlong), respectively. PCR parameters for production of libraryfragments were 5 min initial denaturation at 94° C., 25 cycles of 1 min94° C., 1 min 58° C., 1 min 72° C. and terminal elongation for 10 min at72° C. For assembly PCR, using equimolar ratios of the gel-purifiedsingle fragments as template, parameters were 3 min initial denaturationat 94° C. and 5 cycles of 30 s 94° C., 1 min 58° C., 2 min 72° C. Atthis stage, outer primers (LMB3 and fdseqlong) were added and additional20 cycles were performed prior to a terminal elongation for 10 min at72° C. After assembly of sufficient amounts of full length randomizedFab constructs, they were digested NcoI/NheI and ligated into similarlytreated acceptor phagemid vector. Purified ligations were used for ˜60transformations into electrocompetent E. coli TG1. Phagemid particlesdisplaying the Fab library were rescued and purified by PEG/NaClpurification to be used for selections. A final library size of 9.5×10⁹was obtained. Percentages of functional clones, as determined byC-terminal tag detection in dot blot, were 81.1% for the light chain and83.2% for the heavy chain, respectively.

Table 38 shows the sequence of generic phage-displayed antibody library(DP88-4), Table 39 provides cDNA and amino acid sequences of libraryDP88-4 germline template and Table 40 shows the Primer sequences usedfor generation of DP88-4 germline template.

TABLE 38 Sequence of generic phage-displayed antibody library (DP88-4)SEQ ID NO: Description Sequence 132 nucleotideTGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCG sequence ofCGGCCCAGCCGGCCATGGCCGACATCCAGATGACCCAGTCTCCT pRJH33TCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGC libraryCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCA templateGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCA DP88-4GTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCC library;GGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGA completeTTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTACGTT Fab codingTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCA regionCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT comprisingGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA PelB leaderGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG sequence +GTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG Vk1_5CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT kappa V-ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG domain +CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTG CL constantGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGG domain forAGCCGCAGACTACAAGGACGACGACGACAAGGGTGCCGCATAA light chainTAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATA and PelB +CCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCA VH1_69 V-GCCGGCGATGGCCCAGGTGCAATTGGTGCAGTCTGGGGCTGAGG domain +TGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC CH1GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGC constantCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCT domain forTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACC heavy chainATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAG includingCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAC tagsTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCGCGGCA

TABLE 39cDNA and amino acid sequences of library DP88-4 germline template SEQ IDNO: Description Sequence 133 nucleotideGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC sequence of FabATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCA light chain Vk1_5GTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGG TGCCGCA 134 Fab light chainDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP Vk1_5GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGAAEQKLISEEDLNGAADYKDDDDK GAA 135 nucleotideCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA sequence of FabAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC heavy chainGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC VH1_69GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCG CGGCA 136 Fab heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VH1_69APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDAAASTSAHHHH HHAAA

TABLE 40 Primer sequences used for generation of DP88-4 library SEQID NO: Primer name Primer sequence 5′-3′ 137 LMB3CAGGAAACAGCTATGACCATGATTAC 138 Vk1_5_L3r_S CTCGACTTTGGTGCCCTGGCCAAACGTS

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCATunderlined: 60% original base and 40% randomization as M.bolded and italic: 60% original base and 40% randomization as N 139Vk1_5_L3r_SY CTCGACTTTGGTGCCCTGGCCAAACGTM

S

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCATunderlined: 60% original base and 40% randomization as M.bolded and italic: 60% original base and 40% randomization as N 140Vk1_5_L3r_SPY CTCGACTTTGGTGCCCTGGCCAAACGTM

M SS S

A

C

A

A

CTGTTGGCAGTAATAAGTTGCAAAATCATunderlined: 60% original base and 40% randomization as M.bolded and italic: 60% original base and 40% randomization as N 141RJH31 ACGTTTGGCCAGGGCACCAAAGTCGAG 142 RJH32 TCTCGCACAGTAATACACGGCGGTGTCC143 DP88-v4-4 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S =10%, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E =4.6%; 3: G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%.144 DP88-v4-6 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S =10%, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E =4.6%; 3: G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%.145 DP88-v4-8 GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC 1: G/D = 20%, E/V/S =10%, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E =4.6%; 3: G/A/Y = 20%, P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%.146 fdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG

Table 41 shows the sequence of generic phage-displayed lambda-DP47library (Vl3_19/VH3_23) template used for PCRs. Table 42 provides cDNAand amino acid sequences of lambda-DP47 library (Vl3_19/VH3_23) germlinetemplate and Table 43 shows the Primer sequences used for generation oflambda-DP47 library (Vl3_19/VH3_23).

TABLE 41 Sequence of generic phage- displayed lambda-DP47 library(Vl3_19/VH3_23) template used for PCRs SEQ ID NO: Description Sequence158 pRJH53 ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTC libraryGCGGCCCAGCCGGCCATGGCCTCGTCTGAGCTGACTCAGGACCC template ofTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCC lambda-AAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAG DP47AAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAA libraryCCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAG Vl3_19/VH3_23;GAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT completeGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGCGGTAATCA Fab codingTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAAC regionCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAG comprisingGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGA PelB leaderCTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCA sequence +GCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCA Vl3_19GAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACC lambda V-CCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGA domain +CCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGA CL constantGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGAT domain forCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTG light chainCCGCATAATAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCAT and PelB +ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTC VH3_23_V-GCTGCCCAGCCGGCGATGGCCGAGGTGCAATTGCTGGAGTCTGG domain +GGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CH1CAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCC constantGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGT domain forGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCG heavy chainGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGC includingAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGT tagsGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCA TCACCATCACCATCACGCCGCGGCA

TABLE 42 cDNA and amino acid sequences of lambda-DP47 library(Vl3_19/VH3_23) germline template SEQ ID NO: Description Sequence 159nucleotide TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCT sequence of FabTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCC light chain Vl3_19TCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGCGGTAATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGAC GACGACAAGGGTGCCGCA 160Fab light chain SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQ Vl3_19APVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGAAEQKLISEEDLNGAADYKDDDDKGAA 150 nucleotide see Table 7sequence of Fab heavy chain VH3_23 151 Fab heavy chain see Table 7VH3_23 (DP47)

TABLE 43 Primer sequences used for generation of lambda-DP47 library(V13_19/VH3_23) SEQ ID NO: Primer name Primer sequence 5′-3′ 161 LMB3CAGGAAACAGCTATGACCATGATTAC 162 V1_3_19_L3r_VGGACGGTCAGCTTGGTCCCTCCGCCGAATAC V

 A

 A

G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGCunderlined: 60% original base and 40% randomization as Mbold and italic: 60% original base and 40% randomization as N 163V1_3_19_L3r_HV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC C

 A

A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTCCGCunderlined: 60% original base and 40% randomization as Mbolded and italic: 60% original base and 40% randomization as N 164V1_3_19_L3r_HLV GGACGGTCAGCTTGGTCCCTCCGCCGAATAC R

 V

A

 A

 A

 G

 A

 A

 A

GGAGTTACAGTAATAGTCAGCCTCATCTTC CGCunderlined: 60% original base and 40% randomization as Mbolded and italic: 60% original base and 40% randomization as N 165RJH80 TTCGGCGGAGGGACCAAGCTGACCGTCC 248 MS63 TTTCGCACAGTAATATACGGCCGTGTCC154 DP47-v4-4 CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 155 DP47-v4-6CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 156 DP47-v4-8CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-2-2-3-4-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG 157 fdseqlongGACGTTAGTAAATGAATTTTCTGTATGAGG 1: G/D = 20%, E/V/S = 10%, A/P/R/L/T/Y =5%; 2: G/Y/S = 15%, A/D/T/R/P/L/V/N/W/F/I/E = 4,6%; 3: G/A/Y = 20%,P/W/S/D/T = 8%; 4: F = 46%, L/M = 15%, G/I/Y = 8%; 5: K = 70%, R = 30%.

Human, murine and cynomolgus 4-1BB (CD137) as antigens for the phagedisplay selections and ELISA- and SPR-based screenings were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located a the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain).

Selection rounds (biopanning) were performed in solution according tothe following procedure. First step, pre-clearing of ˜10¹² phagemidparticles on maxisorp plates coated with 10 ug/ml of an unrelated humanIgG to deplete the libraries of antibodies recognizing the Fc-portion ofthe antigen; second, incubation of the non-binding phagemid particleswith 100 nM biotinylated human or murine 4-1BB for 0.5 h in the presenceof 100 nM unrelated non-biotinylated Fc knob-into-hole construct forfurther depletion of Fc-binders in a total volume of 1 ml; third,capture of biotinylated hu 4-1BB and attached specifically binding phageby transfer to 4 wells of a neutravidin pre-coated microtiter plate for10 min (in rounds 1 & 3); fourth, washing of respective wells using5×PBS/Tween20 and 5×PBS; fifth, elution of phage particles by additionof 250 ul 100 mM TEA (triethylamine) per well for 10 min andneutralization by addition of 500 ul 1M Tris/HCl pH 7.4 to the pooledeluates from 4 wells; sixth, post-clearing of neutralized eluates byincubation on neutravidin pre-coated microtiter plate with 100 nMbiotin-captured Fc knob-into-hole construct for final removal ofFc-binders; seventh, re-infection of log-phase E. coli TG1 cells withthe supernatant of eluted phage particles, infection with helperphageVCSM13, incubation on a shaker at 30° C. over night and subsequentPEG/NaCl precipitation of phagemid particles to be used in the nextselection round. Selections were carried out over 3 or 4 rounds usingconstant antigen concentrations of 100 nM. In rounds 2 and 4, in orderto avoid enrichment of binders to neutravidin, capture of antigen: phagecomplexes was performed by addition of 5.4×10⁷ streptavidin-coatedmagnetic beads. Specific binders were identified by ELISA as follows:100 ul of 25 nM biotinylated human or murine 4-1BB and 10 ug/ml of humanIgG were coated on neutravidin plates and maxisorp plates, respectively.Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human or murine 4-1BB and being negative onhuman IgG were short-listed for further analyses and were also tested ina similar fashion against the remaining two species of 4-1BB. They werebacterially expressed in a 0.5 liter culture volume, affinity purifiedand further characterized by SPR-analysis using BioRad's ProteOn XPR36biosensor.

Clones 12B3, 25G7, 11D5 and 9B11 were identified as human 4-1BB-specificbinder through the procedure described above. Clone 20G2 was identifiedas murine 4-1BB-specific binder through the procedure described above.The cDNA sequences of their variable regions are shown in Table 44below, the corresponding amino acid sequences can be found in Table C.

TABLE 44 Variable region base pair sequences for phage-derivedanti-4-1BB antibodies. Underlined are the complementarity determiningregions (CDRs). SEQ ID Clone NO: Sequence 12B3 249 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATCATTCGTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 250 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 25G7 251(VL)TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCTTGATAGGCGCGGTATGTGGGTATTCGGCGGAGGGACCAAGCTGACCGTC 252 (VH)GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 11D5 253 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCTTAATTCGTATCCTCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 254 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 9B11 255 (VL)GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGTTAATTCTTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 256 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 20G2 257 (VL)GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCAGCACTCGTATTATACGTTTGGCCAGGGCACCAAAGTCGAGATCAAG 258 (VH)CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTACTACTGGGAATCTTACCCGTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCAGC6.3 Preparation, Purification and Characterization of Anti-4-1BB IgG1P329G LALA Antibodies

The variable regions of heavy and light chain DNA sequences of selectedanti-4-1BB binders were subcloned in frame with either the constantheavy chain or the constant light chain of human IgG1. The Pro329Gly,Leu234Ala and Leu235Ala mutations have been introduced in the constantregion of the knob and hole heavy chains to abrogate binding to Fc gammareceptors according to the method described in International PatentAppl. Publ. No. WO 2012/130831 A1.

The nucleotide and amino acid sequences of the anti-4-1BB clones areshown in Table 45. All anti-4-1BB-Fc-fusion encoding sequences werecloned into a plasmid vector, which drives expression of the insert froman MPSV promoter and contains a synthetic polyA signal sequence locatedat the 3′ end of the CDS. In addition, the vector contains an EBV OriPsequence for episomal maintenance of the plasmid.

TABLE 45 Sequences of anti-4-1BB clones in P329GLALA human IgG1 formatClone SEQ ID No. Sequence 12B3 259GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATCATTCGTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 260CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA 261DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPG Light chain)KAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 262QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP (Heavy chain)GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 25G7 263TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGG (nucleotideACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAG sequence lightTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCT chain)GTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCTTGATAGGCGCGGTATGTGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 264GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG (nucleotideGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTT sequence heavyAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG chain)GGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 265SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPV (Light chain)LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSLDRRGMWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 266EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG (Heavy chain)LEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 11D5 267GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCTTAATTCGTATCCTCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 268CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 269DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLNSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 270QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9B11 271GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGTTAATTCTTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 272CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA 273DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQVNSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 274QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 20G2 275GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTGT (nucleotideAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATT sequence lightAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCC chain)CCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCAGCACTCGTATTATACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 276CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG (nucleotideGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATT sequence heavyCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAA chain)GGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTACTACTGGGAATCTTACCCGTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCTCCGGGTAAA 277DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPK (Light chain)LLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQQHSYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 278QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG (Heavy chain)LEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSYYWESYPFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

The anti-4-BB antibodies were produced by co-transfecting HEK293-EBNAcells with the mammalian expression vectors using polyethylenimine. Thecells were transfected with the corresponding expression vectors in a1:1 ratio (“vector heavy chain”:“vector light chain”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes at 210×g, and the supernatant was replaced bypre-warmed CD CHO medium. Expression vectors (200 μg of total DNA) weremixed in 20 mL CD CHO medium. After addition of 540 μL PEI, the solutionwas vortexed for 15 seconds and incubated for 10 minutes at roomtemperature. Afterwards, cells were mixed with the DNA/PEI solution,transferred to a 500 mL shake flask and incubated for 3 hours at 37° C.in an incubator with a 5% CO2 atmosphere. After the incubation, 160 mLof F17 medium was added and cells were cultured for 24 hours. One dayafter transfection 1 mM valproic acid and 7% Feed with supplements wereadded. After culturing for 7 days, the supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of antibody molecules from cell culture supernatants wascarried out by affinity chromatography using Protein A as describedabove for purification of antigen Fc fusions.

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl solution of pH 6.0.

The protein concentration of purified antibodies was determined bymeasuring the OD at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the antibodies were analyzed by CE-SDS in the presence andabsence of a reducing agent (Invitrogen, USA) using a LabChipGXII(Caliper). The aggregate content of antibody samples was analyzed usinga TSKgel G3000 SW XL analytical size-exclusion column (Tosoh)equilibrated in a 25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at 25° C.

Table 46 summarizes the yield and final content of the anti-4-BB P329GLALA IgG1 antibodies.

TABLE 46 Biochemical analysis of anti-4-BB P329G LALA IgG1 clones YieldMonomer Clone [mg/l] [%] CE-SDS (non red) CE-SDS (red) 12B3 P329GLALA 498 98.6% (173 kDa) 22.5% (29 kDa) IgG1 75.5% (64 kDa) 25G7 P329GLALA 25100 99.7% (181.6 kDa) 76.8% (65 kDa) IgG1 23% (42 kDa) 11D5 P329GLALA9.7 98.7 99.6% (176 kDa) tbd. IgG1 9B11 P329GLALA 22 100 2% (127 kDa)IgG1 100% (153 kDa) 72.3% (114 kDa) 24.6% (37.1 kDa) 20G2 P329GLALA 11100 98.5% (166 kDa) 80.2% (62.8 kDa) IgG1 18% (28.4 kDa)

Example 7 Characterization of Anti-4-BB Antibodies

7.1 Binding on Human 4-1BB

7.1.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of phage-derived 4-1BB-specific antibodies to the recombinant4-1BB Fc(kih) was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 at 25° C. with HBS-EP asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%Surfactant P20, Biacore, Freiburg/Germany).

In the same experiment, the species selectivity and the avidity of theinteraction between the phage display derived anti-4-1BB clones 12B3,25G7, 11D5, 9B11 and 20G2 (all human IgG1 P329GLALA), and recombinant4-1BB (human, cyno and murine) was determined. Biotinylated human,cynomolgus and murine 4-1BB Fc(kih) were directly coupled to differentflow cells of a streptavidin (SA) sensor chip. Immobilization levels upto 100 resonance units (RU) were used. Phage display derived anti-4-1BBhuman IgG1 P329GLALA antibodies were passed at a concentration rangefrom 4 to 450 nM (3-fold dilution) with a flow of 30 μL/minute throughthe flow cells over 120 seconds. Complex dissociation was monitored for220 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained in a reference flow cell, where noprotein was immobilized.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration and used to estimatequalitatively the avidity (Table 47).

In the same experiment, the affinities of the interaction between phagedisplay derived antibodies (human IgG1 P329GLALA) to recombinant 4-1BB(human, cyno and murine) were determined. Anti-human Fab antibody(Biacore, Freiburg/Germany) was directly coupled on a CM5 chip at pH 5.0using the standard amine coupling kit (Biacore, Freiburg/Germany). Theimmobilization level was approximately 7500 RU. Phage display derivedantibodies to 4-1BB were captured for 60 seconds at concentrationsranging from 25 nM. Recombinant human 4-1BB Fc(kih) was passed at aconcentration range from 4.1 to 1000 nM with a flow of 30 μL/minutesthrough the flow cells over 120 seconds. The dissociation was monitoredfor 120 seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantigens were flown over a surface with immobilized anti-human Fabantibody but on which HBS-EP has been injected rather than theantibodies. Kinetic constants were derived using the Biacore T200Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rateequations for 1:1 Langmuir binding by numerical integration.

Clones 25G7 and 9B11 bind human 4-1BB Fc(kih) with a lower affinity thanclones 12B3 and 11D5. Clone 20G2 is not binding to human 4-1BB. Affinityconstants for the interaction between anti-4-1BB P329GLALA IgG1 andhuman 4-1BB Fc(kih) were determined by fitting to a 1:1 Langmuirbinding.

TABLE 47 Binding of anti-4-1BB antibodies to recombinant human 4-1BBRecombinant human 4-1BB Recombinant (affinity format) human 4-1BB ka kdKD Clone Origin (avidity format) (1/Ms) (1/s) (M) 12B3 Phage ++++3.4E+04 1.0E−03 3.0E−08 display 25G7 Phage + 2.9E+04 9.9E−04 3.4E−08display 11D5 Phage +++ 3.2E+04 1.2E−03 3.6E−08 display 9B11 Phage ++2.7E+04 3.9E−03 1.4E−07 display7.1.2 Binding to Human 4-1BB Expressing Cells: Resting and ActivatedHuman Peripheral Mononuclear Blood Leukocytes (PBMC)

Expression of human 4-1BB is absent on resting and naïve human T cells(Kienzle G. and von Kempis J (2000), Int. Immunol. 12(1), 73-82, Wen T.et al. (2002), J. Immunol. 168, 4897-4906). After activation withimmobilized anti-human CD3 agonistic antibody, 4-1BB is upregulated onCD4⁺ and CD8⁺ T cells. 4-1BB expression has also been reported onactivated human NK cells (Baessler T. et. al. (2010) Blood 115(15),3058-3069), activated human NKT cells (Cole S. L. et al. (2014) J.Immunol. 192(8), 3898-3907), activated human B cells (Zhang et al.(2010) J. Immunol. 184(2), 787-795), activated human eosinophils(Heinisch et al. 2001), constitutively on human neutrophils (Heinisch I.V. (2000) J Allergy Clin Immunol. 108(1), 21-28), activated humanmonocytes (Langstein J. et al. (1998) J Immunol. 160(5), 2488-2494,Kwajah M. and Schwarz H. (2010) Eur J Immunol. 40(7), 1938-1949),constitutively on human regulatory T cells (Bacher P. et al. (2014)Mucosal Immunol. 7(4), 916-928), human follicular dendritic cells (PaulyS. et al. (2002) J Leukoc Biol. 72(1), 35-42), activated human dendriticcells (Zhang L. et al. (2004) Cell Mol Immunol. 1(1), 71-76) and onblood vessels of malignant human tumors (Broll K. et al. (2001) Am JClin Pathol. 115(4), 543-549).

To test binding of our anti-4-1BB clones to naturally cell-expressedhuman 4-1BB, fresh isolated resting peripheral blood mononuclear cells(PBMCs) or PHA-L/Proleukin pre-activated and CD3/CD28-reactivated PBMCwere used. PBMCs from buffy coats obtained from the Zurich blooddonation center were isolated by ficoll density centrifugation usingHistopaque 1077 (SIGMA Life Science, Cat.-No. 10771, polysucrose andsodium diatrizoate, adjusted to a density of 1.077 g/mL) and resuspendedin T cell medium consisting of RPMI 1640 medium (Gibco by LifeTechnology, Cat.-No. 42401-042) supplied with 10% Fetal Bovine Serum(FBS, Gibco by Life Technology, Cat.-No. 16000-044, Lot 941273,gamma-irradiated, mycoplasma-free and heat inactivated at 56° C. for 35min), 1% (v/v) GlutaMAX-I (GIBCO by Life Technologies, Cat.-No. 35050038), 1 mM Sodium Pyruvate (SIGMA, Cat.-No. S8636), 1% (v/v) MEMnon-essential amino acids (SIGMA, Cat.-No. M7145) and 50 μMβ-Mercaptoethanol (SIGMA, M3148). PBMCs were used directly afterisolation (resting cells) or stimulated to induce 4-1BB expression atthe cell surface of T cells by culturing for 3 to 5 days in T cellmedium supplemented with 200 U/mL Proleukin (Novartis Pharma Schweiz AG,CHCLB-P-476-700-10340) and 2 μg/mL PHA-L (SIGMA Cat.-No. L2769) in a6-well tissue culture plate and then 2 day in a 6-well tissue cultureplate coated with 10 μg/mL anti-human CD3 (clone OKT3, BioLegend,Cat.-No. 317315) and 2 μg/mL anti-human CD28 (clone CD28.2, BioLegend,Cat.-No.: 302928) in T cell medium at 37° C. and 5% CO₂.

To determine binding to human 4-1BB expressed by human PBMCs,0.1-0.2×10⁶ freshly isolated or activated PBMCs were added to each wellof a round-bottom suspension cell 96-well plates (Greiner bio-one,cellstar, Cat.-No. 650185). Plates were centrifuged 4 minutes with 400×gat 4° C. and supernatant was discarded. Cells were washed with 200μL/well DPBS and then incubated for 30 min at 4° C. with 100 μL/mL DPBScontaining 1:5000 diluted Fixable Viability Dye eFluor 450 (eBioscience,Cat.-No. 64-0863-18) or Fixable Viability Dye eFluor 660 (eBioscience,Cat.-No. 65-0864-18). Afterwards cells were washed once with 200 μL/wellcold FACS buffer (DPBS supplied with 2% (v/v) FBS, 5 mM EDTA pH8(Amresco, Cat. No. E177) and 7.5 mM sodium azide (Sigma-Aldrich S2002)).Next, 50 μL/well of 4° C. cold FACS buffer containing titratedanti-human 4-1BB binders were added and cells were incubated for 120minutes at 4° C. Cells were washed four times with 200 μL/well 4° C.FACS buffer to remove onbound molecules. Afterwards cells were furtherincubated with 50 μL/well of 4° C. cold FACS buffer containing 2.5 μg/mLPE-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatF(ab′)2 fragment (Jackson ImmunoResearch, Cat.-No. 109-116-098) or 30μg/mL FITC-conjugated AffiniPure anti-human IgG Fcγ-fragment-specificgoat F(ab′)2 fragment (Jackson ImmunoResearch, Cat.-No. 109 096 098),anti-human CD45 AF488 (clone HI30, BioLegend, Cat.-No. 304019), 0.67μg/mL APC/Cy7-conjugated anti-human CD3 moIgG1κ (clone UCH1, BioLegend,Cat.-No. 300426) or 0.125 μg/mL PE-conjugated anti-human CD3 mouse IgG1κ(clone SK7, BioLegend Cat.-No. 344806) or 0.67 μg/mLPerCP/Cy5.5-conjugated anti-human CD3 mouse IgG1 κ (clone UCHT1,BioLegend, Cat.-No. 300430), 0.125 μg/mL BV421-conjugated anti-human CD4moIgG1κ (clone RPA-T4, BioLegend, Cat.-No. 300532) or 0.23 μg/mLBV421-conjugated anti-human CD4 mouse IgG2b κ (clone OKT4, BioLegend,Cat.-No. 317434) or 0.08 μg/mL PE/Cy7-conjugated anti-human CD4 mouseIgG1κ (clone SK3, BioLegend Cat.-No. 344612), 0.17 μg/mLAPC/Cy7-conjugated anti-human CD8 (mouse IgG1κ, clone RPA-T8, BioLegendCat.-No. 301016) or 0.125 μg/mL PE/Cy7-conjugated anti-human CD8a(moIgG1κ, clone RPA-T8, BioLegend, Cat.-No. 301012) or 0.33 μg/mLanti-human CD8 BV510 (moIgG1κ, clone SK1, BioLegend, Cat.-No. 344732)and 0.25 μg/mL APC-conjugated anti-human CD56 (mouse IgG1κ, clone HCD56,BioLegend, Cat.-No. 318310) or 1 μL AF488-conjugated anti-human CD56(moIgG1κ, clone B159, BD Pharmingen, Cat.-No. 557699) and 0.67 μg/mLanti-human CD19-PE/Cy7 (moIgG1κ, clone HIB19, BioLegend, Cat.-No.302216) and incubated for 30 minutes at 4° C.

Cells were washed twice with 200 μL FACS buffer/well and fixated byresuspending in 50 μL/well DPBS containing 1% Formaldehyde (Sigma,HT501320-9.5L). Cells were acquired the same or next day using a 3-laserCanto II (BD Bioscience with DIVA software) or a 5-laser Fortessa (BDBioscience with DIVA software) or 3-laser MACSQuant Analyzer 10(Miltenyi Biotech). Gates were set on CD8⁺ and CD4⁺ T cells and themedian fluorescence intensity (MFI) or geo mean of fluorescenceintensity of the secondary detection antibody was used to analyzebinding of primary antibodies. Using Graph Pad Prism (Graph Pad SoftwareInc.) data was baselined by subtracting the blank values (no primaryantibody added) and the EC50 values were calculated using non-linearregression curve fit (robust fit).

Human T cells lack 4-1BB expression in a resting status but upregulate4-1BB after activation. Human CD8⁺ T cells show a stronger up-regulationthan CD4⁺ T. The generated anti-human 4-1BB-specific antibodies can bindto human 4-1BB expressed by activated human T cells as shown in FIGS.23A-23D. The shown anti-human 4-1BB clones can be classified in strongbinding (clones 12B3 and 11D5) and low binders (clones 25G7 and 9B11).Differences are not only seen by EC₅₀ value but also by MFI. The EC₅₀values of binding to activated CD8 T cells are shown in Table 48. Theanti-mouse 4-1BB-specific clone 20G2 did not bind to human 4-1BB and istherefore not human-cross-reactive.

TABLE 48 EC₅₀ values of binding to activated human CD8 T cells CloneEC₅₀ [nM] 25G7 29 12B3 0.95 11D5 1.46 9B11 4.485 20G2 n.d.7.2 Binding on Murine 4-1BB7.2.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of the phage-derived 4-1BB specific antibody 20G2 to recombinantmurine 4-1BB Fc(kih) was assessed by surface plasmon resonance asdescribed above for human 4-1BB Fc(kih) (see Example 7.1.1). Kineticconstants were derived using the Biacore T200 Evaluation Software (vAA,Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuirbinding by numerical integration and used to estimate qualitatively theavidity (Table 44).

For affinity determination, due to an unspecific interaction of the Fcfusion protein to the reference flow cell, murine 4-1BB Fc(kih) wascleaved with AcTEV protease and the Fc portion removed bychromatographical method. Anti-human Fc antibody (Biacore,Freiburg/Germany) was directly coupled on a CM5 chip at pH 5.0 using thestandard amine coupling kit (Biacore, Freiburg/Germany). Theimmobilization level was about 7500 RU. Phage display derived antibodiesto 4-1BB were captured for 60 seconds at concentrations ranging from 25nM. Recombinant murine 4-1BB AcTEV was passed at a concentration rangefrom 4.1 to 1000 nM with a flow of 30 μL/minutes through the flow cellsover 120 seconds. The dissociation was monitored for 120 seconds. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on reference flow cell. Here, the antigens were flownover a surface with immobilized anti-human Fc antibody but on whichHBS-EP has been injected rather than the antibodies.

Kinetic constants were derived using the Biacore T200 EvaluationSoftware (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for1:1 Langmuir binding by numerical integration. It was shown that clone20G2 binds murine 4-1BB (Table 49).

Affinity constants of interaction between anti-4-1BB P329GLALA IgG1molecules and murine 4-1BB were derived using the Biacore T200Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rateequations for 1:1 Langmuir binding by numerical integration.

TABLE 49 Binding of anti-4-1BB antibody 20G2 to murine 4-1BB Recombinantmurine 4-1BB Recombinant (affinity format) murine 4-1BB ka kd KD CloneOrigin (avidity format) (1/Ms) (1/s) (M) 20G2 Phage +++++ 2.4E+043.4E−04 1.4E−08 display7.2.2 Binding to Mouse 4-1BB Expressing Cells: Resting and ActivatedMouse Splenocytes (Selected Clones)

Similar to human, freshly isolated resting mouse T cells do not express4-1BB but expression can be induced by TCR activation via peptide-pulsedAPCs (Cannons J. et al. (2001) J. Immunol. 167(3): 1313-1324) orimmobilized anti-mouse CD3 or a combination of anti-mouse CD3 andanti-mouse CD28 antibodies (Pollok K. et al. (1995), European J.Immunol. 25(2), 488-494). After activation via surface immobilizedanti-CD3 antibody 4-1BB expression is reported to be higher on CD8⁺ Tcells (Shuford W, et al. (1997) J. of Experimental Med. 186(1), 47-55),but this may depend on activation protocol. Further 4-1BB expression hasalso been reported on activated mouse NK cells (Melero et al. (1998)Cell Immunol. 190(2), 167-172), activated mouse NKT cells (Vinay D, etal. (2004) J Immunol. 173(6), 4218-4229), activated mouse B cells(Vinay, Kwon (2011) Cell Mol Immunol. 8(4), 281-284), constitutively onmouse neutrophils (Lee S. et al. (2005) Infect Immun. 73(8), 5144-5151)and mouse regulatory T cells (Gavin et al. (2002) Nat Immunol. 3(1),33-41), mouse IgE-stimulated mast cell (Nishimoto et al. (2005) Blood106(13): 4241-4248), mouse myeloid-lineage cells (Lee et al. (2008) NatImmunol. 9(8), 917-926), mouse follicular dendritic cells (Middendorp etal. (2009) Blood 114(11), 2280-2289) and activated mouse dendritic cells(Wilcox, Chapoval et al. (2002) J Immunol. 168(9),4262-4267).

Anti-4-1BB specific antibody binding was tested directly after isolationof splenocytes from healthy female C57BL/6 mice as well as after invitro activation for 72 hours with surface immobilized agonisticanti-mouse CD3 and anti-mouse CD28 antibodies. Female C57BL/6 mice (age7-9 weeks) were purchased at Charles River, France. After arrivalanimals were maintained for one week to get accustomed to newenvironment and for observation. Mice were maintained underspecific-pathogen-free condition with food and water ad libitum anddaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). Continuous health monitoringwas carried out on a regular basis. Mice were sacrificed by cervicaldislocation. Spleens were dissected and stored on ice in RPMI 1640supplemented with 10% (v/v) heat-inactivated FBS and 1% (v/v)GlutaMAX-I. To obtain a single cell solution spleens were homogenizedthrough a 70 μm cell strainer (BD Falcon; Germany) and subjected toerythrolysis for 10 minutes at 37° C. in ACK lysis buffer (0.15M NH4CL,10 mM KHCO3, 0.1 mM EDTA in ddH₂O, pH 7.2). After two washing steps withsterile DPBS splenocytes were reconstituted in T cell medium. Eithercells were used freshly (resting) or 10⁶ cells/mL splenocytes werefurther stimulated for 72 hours in T cell medium consisting of RPMI 1640medium supplied with 10% FBS, 1% (v/v) GlutaMAX-I, 1 mM Sodium Pyruvate,1% (v/v) MEM non-essential amino acids and 50 μM β-Mercaptoethanol on6-well cell culture plates coated with 1 μg/mL anti-mouse CD3 antibody(rat IgG2b, clone 17A2, BioLegend, Cat.-No. 100223) and 2 μg/mLanti-mouse CD28 antibody (syrian hamster, clone 37.51, BioLegendCat.-No. 102112).

To test binding to mouse 4-1BB, freshly isolated or activated mousesplenocytes were resuspended in DPBS and 0.1×10⁶/well splenocytes weretransferred to a round-bottom 96-suspension cell plate (Greiner bio-one,cellstar, Cat.-No. 650185). Cells were centrifuged 4 minutes at 4° C.and 400×g and supernatant was removed. After resuspension in 100μ/wellDPBS containing 1:5000 diluted Fixable Viability Dye eFluor 450(eBioscience, Cat.-No. 65-0863-18) or Fixable Viability Dye eFluor 660(eBioscience, Cat.-No. 65-0864-18) cells were incubated for 30 min at 4°C. Cells were washed with FACS buffer and 50 μL/well FACS buffercontaining titrated concentrations of anti-human 4-1BB huIgG1 P329G LALAantibody-clones 12B3, 25G7, 11D5, 9B11 and anti-mouse 4-1BB-specificclone 20G2 as huIgG1 P329G LALA or mouse IgG1 or mouse IgG1 DAPG. After1 h incubation at 4° C. cells were washed four times to remove excessiveantibodies. If binding of anti-mouse 20G2 as mouse IgG1 and mouse IgG1DAPG format was tested, cells were incubated in 50 μL FACS buffer/wellcontaining 30 μg/mL FITC-conjugated anti-mouse IgG Fcγ-fragment-specificAffiniPure goat F(ab′)γ fragment for 30 min at 4° C. and washed twicewith FACS-buffer. If binding of anti-4-1BB binders containing a humanIgG1 P329G LALA Fc-fragment were tested this step was skipped.Afterwards cells were incubated in 50 μL FACS buffer/well containing0.67 μg/mL PE-conjugated anti-mouse CD3 (rat IgG2bκ, clone 17A2, BDPharmingen, Cat.-No. 555275) or APC-Cy7-conjugated anti-mouse CD3 (ratIgG2aκ, clone 53-6.7, BioLegend, Cat.-No. 100708), 0.67 μg/mLPE/Cy7-conjugated anti-mouse CD4 (rat IgG2bκ, clone GK1.5, BioLegend,Cat.-No. 100422), 0.67 μg/mL APC/Cy7-conjugated anti-mouse CD8 (ratIgG2aκ, clone 53-6.7, BioLegend, Cat.-No. 1007141) or PE-conjugatedanti-mouse CD8 (rat IgG2aκ, clone 53-6.7, BioLegend, Cat.-No. 100708), 2μg/mL APC-conjugated anti-mouse NK1.1 (mouse IgG2a, κ, clone PK136,BioLegend, Cat.-No. 108710) or PerCP/Cy5.5-conjugated anti-mouse NK1.1(mouse IgG2a, κ, clone PK136, BioLegend, Cat.-No. 108728) and 10 μg/mLanti-mouse CD16/CD32 (mouse Fc-Block, rat IgG2b κ, clone 2.4G2, BDBioscience, Cat.-No. 553142). If binding of anti-4-1BB binderscontaining a human IgG1 P329G LALA Fc-fragment were tested 30 μg/mLFITC-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatF(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109 096 098) werealso added. Cells were incubated for 30 min at 4° C., washed twice with200 μL/well FACS-buffer and resuspended for fixation with 50 μL/wellDPBS containing 1% (v/v) formaldehyde. The next day cells wereresuspended in 200 μL/well FACS buffer and acquired using the 2-laserCantoII (BD Bioscience with DIVA software) or 5-laser Fortessa (BDBioscience with DIVA software). Gates were set on CD8⁺ and CD4⁺ T cellsand the median fluorescence intensity (MFI) of the secondary detectionantibody was used to analyze binding of primary antibodies. Using GraphPad Prism (Graph Pad Software Inc.) data was baselined by subtractingthe blank values (no primary antibody added) and the EC₅₀ values werecalculated using non-linear regression curve fit (robust fit).

As shown in FIGS. 24B and 24D, only the anti-mouse 4-1BB binding clone20G2 bound to activated mouse CD8⁺ and CD4⁺ T cells, whereas theanti-human-4-1BB binding clones 9B11, 11D5, 12B3 and 25G7 did not bindto mouse-4-1BB and are therefore not mouse-cross-reactive. Only clone20G2 from the tested clones can be used as a mouse surrogate. Asexpected none of the anti-4-1BB binding clones showed binding to freshlyisolated resting mouse T cells (FIGS. 24A and 24C). Similar to theactivated human CD4⁺ T cells also the activated mouse CD4⁺ T cellsexpress less 4-1BB than the activated mouse CD8⁺ T cells. The differencehowever is not as strong as for human T cells, this may also be relatedto the different activation protocol. EC₅₀ values are shown in Table 50.

TABLE 50 EC₅₀ values of binding to activated murine CD8 and CD4 T cellsClone EC₅₀ CD8 [nM] EC₅₀ CD4 [nM] 25G7 n.d. n.d. 12B3 n.d. n.d. 11D5 n.dn.d 9B11 n.d n.d 20G2 16.36 10.10

As shown in FIGS. 25B and 25D, the anti-mouse-4-1BB binding clone 20G2binds to activated mouse CD8⁺ and CD4⁺ T cells as moIgG1 wildtype (wt)or moIgG1 DAPG format in a similar way. The binding is similar to thebinding shown in FIGS. 24B and 24D; therefore changing of format doesnot influence the binding properties. We transferred the clone 20G2 tomoIgG to prevent triggering of anti-drug-antibodies (ADAs) in immunecompetent mice. The DAPG mutation is equivalent to the P329G LALAmutation in the human IgG1 constructs, e.g. it prevents crosslinking viathe FcR⁺ immune cells. EC50 values are shown in Table 51.

TABLE 51 EC₅₀ values of binding to activated murine CD8 and CD4 T cellsClone EC₅₀ CD8 [nM] EC₅₀ CD4 [nM] 20G2 mu IgG1 κ DAPG 17.91 11.22 20G2mu IgG1 κ wt 18.51 9.9177.3 Binding on Cynomolgus 4-1BB7.3.1 Surface Plasmon Resonance (Avidity+Affinity)

Binding of phage-derived 4-1BB specific antibodies 12B3, 25G7, 11D5 and9B11 to the recombinant cynomolgus 4-1BB Fc(kih) was assessed by surfaceplasmon resonance (SPR) as described above for human 4-1BB Fc(kih) (seeExample 7.1.1). All SPR experiments were performed on a Biacore T200 at25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl,3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany). Kineticconstants were derived using the Biacore T200 Evaluation Software (vAA,Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuirbinding by numerical integration and used to estimate qualitatively theavidity (Table 52).

In the same experiment, the affinities of the interaction between phagedisplay derived antibodies (human IgG1 P329GLALA) to recombinantcynomolgus 4-1BB Fc(kih) were determined. Anti-human Fab antibody(Biacore, Freiburg/Germany) was directly coupled on a CM5 chip at pH 5.0using the standard amine coupling kit (Biacore, Freiburg/Germany). Theimmobilization level was approximately 9000 RU. Phage display derivedantibodies to 4-1BB were captured using concentrations of 25 to 100 nM.The experiment was performed as described for human 4-1BB Fc(kih(Example 7.1.1).

Clones 12B3, 25G7, 11D5 and 9B11 bound cynomolgus 4-1BB Fc(kih) withsimilar affinities (Table 52), but 12B3 and 11D5 bound with higheravidity to cells expressing cynomolgus 4-1BB. Affinity constants ofinteraction between anti-4-1BB P329GLALA IgG1 and cynomolgus 4-1BBFc(kih) were derived using the Biacore T100 Evaluation Software (vAA,Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuirbinding by numerical integration.

TABLE 52 Binding of anti-4-1BB antibodies to recombinant cynomolgus4-1BB Fc(kih) Recombinant cynomolgus 4-1BB Recombinant (affinity format)cynomolgus 4-1BB ka kd KD Clone Origin (avidity format) (1/Ms) (1/s) (M)12B3 Phage +++ 3.8E+04 7.8E−04 2.0E−08 display 25G7 Phage ++ 2.7E+044.6E−04 1.7E−08 display 11D5 Phage +++ 3.1E+04 7.7E−04 2.4E−08 display9B11 Phage + 2.6E+04 2.0E−03 7.8E−08 display7.3.2 Binding on Cynomolgus 4-1BB Expressing Cells: Activated CynomolgusPeripheral Mononuclear Blood Leukocytes (PBMC)

To test the cross-reactivity of the anti-human 4-1BB binding clones tocynomolgus cells, PBMCs of healthy cynomolgus fascicularis were isolatedfrom heparinized blood using density gradient centrifugation asdescribed for human PBMCs (7.1.2) with minor differences. Isolated PBMCwere cultured for 72 hours at a cell density of 1.5*10⁶ cells/mL in Tcell medium consisting of RPMI 1640 medium supplied with 10% FBS, 1%(v/v) GlutaMAX-I, 1 mM Sodium Pyruvate, 1% (v/v) MEM non-essential aminoacids and 50 μM β-Mercaptoethanol on 6-well cell culture plates (GreinerBio-One, Germany) coated with 10 μg/mL anti-cyno-cross-reactive CD3 (moIgG3λ, anti-human CD3, clone SP34, BD Pharmingen, Cat.-No. 556610) and 2μg/mL anti-cyno-cross-reactive CD28 (moIgG1κ, anti-human CD28, cloneCD28.2, BioLegend, Cat.-No. 140786) antibodies. After 72 h stimulationcells were harvested and seeded to a round-bottom suspension cell96-well plate (Greiner bio-one, cellstar, Cat.-No. 650185) at aconcentration of 0.1×10⁶ cells/well. Cells were incubated in 50 μL/wellFACS buffer containing different concentrations of the primaryanti-human 4-1BB-specific huIgG P32G LALA antibodies for 2 h at 4° C.Afterwards cells were washed four times with 200 μL/well FACS buffer andincubated further for 30 min at 4° C. with 50 μL/well FACS buffercontaining 2 μL PE-conjugated anti-cyno-crossreactive CD4 (moIgG2aκ,anti-human CD4, clone M-T477, BD Pharmingen, Cat.-No. 556616), 1 μg/mLPerCP/Cy5.5-conjugated anti-cyno-cross-reactive CD8 (moIgG1κ, anti-humanCD8, clone RPA-T8, BioLegend, Cat.-No. 301032) and 30 μg/mLFITC-conjugated AffiniPure anti-human IgG Fcγ-fragment-specific goatF(ab′)2 fragment (Jackson ImmunoResearch, Cat. No. 109 096 098). Cellswere washed twice with FACS buffer and resuspended in 100 μL/well FACSbuffer supplied with 0.2 μg/mL DAPI to discriminated dead from livingcells. Cells were immediately acquired using a 5-laser Fortessa (BDBioscience with DIVA software). Gates were set on CD8⁺ and CD4⁺ T cellsand the median fluorescence intensity (MFI) of the secondary detectionantibody was used to analyze binding of primary antibodies. Using GraphPad Prism (Graph Pad Software Inc.) data was baselined by subtractingthe blank values (no primary antibody added) and the EC₅₀ values werecalculated using non-linear regression curve fit (robust fit).

As shown in FIGS. 26A and 26B, at least three of the anti-human 4-1BBclones, namely 12B3, 11D5 and 25G7 are also cross-reactive forcynomolgus 4-1BB expressed on activated CD4⁺ and CD8⁺ T cells.Interestingly the binding curves look similar to human 4-1BB, e.g. 12B3and 11D5 show the highest MFI and lowest EC₅₀ values of all testedconstructs, whereas 25G7 is a weaker binder with lower MFI and higherEC₅₀ value (Table 53). The clone 9B11 is only binding at the highestconcentration of 100 nM. This is contrary to binding to human 4-1BBwhere clone 9B11 is superior compared to clone 25G7. Further differencesof 4-1BB expression levels on activated cynomolgus CD4⁺ and CD8⁺ T cellsare similar to activated human T cells e.g. CD4⁺ T cells express muchless 4-1BB than CD8⁺ T cells.

TABLE 53 EC₅₀ values of binding to activated cynomolgus CD8 and CD4 Tcells Clone EC₅₀ CD8 [nM] EC₅₀ CD4 [nM] 25G7 4.52 6.68 12B3 0.47 0.5911D5 1.04 0.97 9B11 n.d. n.d.7.4 Ligand Blocking Property

To determine the capacity of 4-1BB-specific human IgG1 P329GLALAantibody molecules to interfere with 4-1BB/4-1BB-ligand interactionshuman 4-1BB ligand (R&D systems) was used. Similarly, murine 4-1BBligand (R&D systems) was used to assess the ligand blocking property ofthe anti-murine 4-1BB specific IgG1 P329GLALA antibody 20G2.

Human or murine 4-1BB ligand was directly coupled to two flow cells of aCM5 chip at approximately 1500 RU by pH 5.0 using the standard aminecoupling kit (Biacore, Freiburg/Germany). Recombinant human, or murine,4-1BB Fc(kih) was passed on the second flow cell at a concentration of500 nM with a flow of 30 μL/minute over 90 seconds. The dissociation wasomitted and the phage derived anti-4-1BB human IgG1 P329GLALA was passedon both flow cells at a concentration of 200 nM with a flow of 30uL/minute over 90 seconds. The dissociation was monitored for 60seconds. Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantibodies were flown over a surface with immobilized human, or murine,4-1BB ligand but on which HBS-EP has been injected instead ofrecombinant human 4-1BB Fc(kih).

The phage-derived clone 25G7 bound to the complex of human 4-1BB withits 4-1BB ligand (Table 54, FIG. 27A). Thus, this antibody does notcompete with the ligand for binding to human 4-1BB and is thereforetermed “non-ligand blocking”. On the contrary, clones 12B3, 11D5 and9B11 did not bind to human 4-1BB associated with its ligand and aretherefore termed “ligand blocking”. The murine surrogate 20G2 did notbind to murine 4-1BB associated with its ligand and is also termed“ligand blocking”

TABLE 54 Ligand binding property of the anti-4-1BB clones determined bysurface plasmon resonance Second injection (anti- Ligand Clone OriginFirst injection 4-1BB clone) blocking 12B3 Phage human 4-1BB Not bindingYES display Fc(kih) 25G7 Phage human 4-1BB Binding NO display Fc(kih)11D5 Phage human 4-1BB Not binding YES display Fc(kih) 9B11 Phage human4-1BB Not binding YES display Fc(kih) 20G2 Phage murine 4-1BB Notbinding YES display Fc(kih)7.5 Epitope Characterization

The epitope recognized by the phage-derived anti-4-1BB antibody wascharacterized by surface plasmon resonance. First, the ability of theantibodies to compete for binding to human 4-1BB was assessed by surfaceplasmon resonance. Second, the binding of the anti-4-1BB antibodies totwo different domains of human and murine 4-1BB was evaluated by SPR asdescribed herein before. For this purpose hybrid 4-1BB Fc(kih) fusionproteins have been designed. They contain the ectodomains of 4-1BBeither a human or a murine domain. The hybrid 4-1BB variants were fusedto the knob chain of the Fc(kih), similarly to the antigens Fc fusionused for the phage display selection and described above. The aim ofthese experiments was to define an “epitope bin”. Antibodies thatcompete for a similar or an overlapping epitope are not able to bindsimultaneously to 4-1BB and belong to an “epitope bin”. Anti-4-1BBantibodies that can bind simultaneously to 4-1BB do not share anepitope, or part of it, and are therefore grouped into a differentepitope bin.

7.5.1 Competition Binding (SPR)

To analyze competitive binding for human or murine receptor of theanti-4-1BB human IgG1 P329GLALA (FIGS. 28A-28F), the phage derivedanti-4-1BB clones 9B11 and 11D5 were directly coupled to CM5 chip atrespectively 1400 and 2200 RU by pH 5.5 using the standard aminecoupling kit (Biacore, Freiburg/Germany). Recombinant human 4-1BBFc(kih) was injected at a concentration of 200 nM with a flow of 30μL/min over 180 seconds. The dissociation was omitted and a secondanti-4-1BB human IgG1 P329GLALA antibody was passed at a concentrationof 100 nM with a flow of 30 μL/min over 90 seconds. Bulk refractiveindex differences were corrected for by subtracting the responseobtained on reference flow cell.

The SPR experiments were performed on a Biacore T200 at 25° C. withHBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% Surfactant P20, Biacore, Freiburg/Germany). The competitionbinding experiment showed that the phage-derived anti-4-1BB clones 12B3,11D5 and 9B11 shares a different spatial epitope as 25G7, since the twoantibodies can bind simultaneously to human 4-1BB Fc(kih) (Table 55).

TABLE 55 Summary of competition binding experiments Immobilized on FirstSecond injection chip injection 25G7 12B3 9B11 11D5 9B11 Human 4- 1 0 X0 1BB Fc(kih) 11D5 Human 4- 1 0 0 X 1BB Fc(kih) 0 = no binding; 1,binding; X = not determined since the second injection contains the sameantibody as the one immobilized on the chip7.5.2 Binding to Receptor Variants (SPR)

The epitopes recognized by the phage-derived anti-4-1BB clones 12B3,25G7, 11D5, 9B11 and 20G2 have been characterized using hybrid variantsof 4-1BB Fc(kih) fusion molecules containing a combination of human andmurine domains, or a single domain (Table 56). The hybrid 4-1BB werefused to Fc(kih) as described in Example 6.1 for the 4-1BB Fc(kih)antigens. Table 52 contains the sequences of the 4-1BB domains used forthe preparation of hybrid 4-1BB variants.

Construct 1 is composed by the N-terminal portion (termed domain 1) ofmurine 4-1BB fused to the C-terminal portion (termed domain 2) of human4-1BB (Table 56). Construct 2 is composed by the N-terminal portion(termed domain 1) of human 4-1BB fused to the C-terminal portion (termeddomain 2) of murine 4-1BB (Table 57). These hybrid molecules wereprepared since the expression of domain 2 did not lead to a functionalmolecule. The N-terminal portion (domain 1) of human 4-1BB was alsoexpressed as Fc(kih) fusion molecule (construct 3). Expression andpurification of the hybrid 4-1BB Fc(kih) variants was performed asdescribed in Example 6.1.

TABLE 56 Amino acid sequences of hybrid 4-1BB Fc(kih) moleculesDomains fused (SEQ ID NO:) Amino acid sequence Construct 1 mu4-1BB D1/VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNIC hu4-1BB D2 FcRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKD knobCRPGQELTKQGCKDCCFGTFNDQKRGICRPWTNCSLDGK (279)SVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKSGGLNDIFEAQKIEWHEFc hole DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC (128)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNRFTQKSLSLSPGKConstruct 2 hu4-1BB D1/ LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDImu4-1BB D2 Fc CRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCE knobQDCKQGQELTKKGCKTCSLGTFNDQNGTGVCRPWTNCS (280)LDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVLVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGKSGGLNDIFEAQKIEWHEFc hole DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC (128)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNRFTQKSLSLSPGKConstruct 3 hu4-1BB D1 Fc LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDI knobCRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCE (281)QDCKQGQELTKKGCKVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE Fc holeDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC (128)VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNRFTQKSLSLSPGK

TABLE 57 Amino acid sequences of human and murine 4-1BB domainsused to prepare the hybrid constructs Domains fused (SEQ ID NO:)Amino acid sequence Construct 1 Murine 4-1BBVQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPN D1 (N-terminus)CNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQ (282) CTRCEKDCRPGQELTKQGCKHuman 4-1BB DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKER D2 (C-terminus)DVVCGPSPADLSPGASSVTPPAPAREPGHSPQ (283) Construct 2 Human 4-1BBLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR D1 (N-terminus)TCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGA (284) GCSMCEQDCKQGQELTKKGCKMurine 4-1BB TCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTE D2 (C-terminus)KDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVL (285) Construct 3 Human 4-1BBLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR D1 (N-terminus)TCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGA (284) GCSMCEQDCKQGQELTKKGCK

The SPR experiments were performed on a Biacore T200 at 25° C. withHBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% Surfactant P20, Biacore, Freiburg/Germany). Anti-human Fabantibody (Biacore, Freiburg/Germany) was directly coupled on a CM5 chipat pH 5.0 using the standard amine coupling kit (Biacore,Freiburg/Germany). The immobilization level was approximately 8,000 RU.Phage-display derived anti-4-1BB antibodies were captured for 60 secondsat 100 nM. Recombinant hybrid human/mouse 4-1BB Fc(kih) variants werepassed at 200 nM with a flow of 30 μL/minute through the flow cells over120 seconds. The dissociation was monitored for 120 seconds (FIGS.29A-29D).

Human 4-1BB-D1 (construct 3) was directly coupled to CM5 chip atapproximately 1000 RU. Anti-4-1BB antibodies at 200 nM were passed at 30μL/minute for 180 sec and dissociation was monitored for 60 seconds(FIGS. 30A-30D). Bulk refractive index differences were corrected for bysubtracting the response obtained on reference flow cell. Here, theantigens were flown over a surface with immobilized anti-human Fabantibody but on which HBS-EP has been injected rather than theantibodies.

The phage-derived clones 12B3, 11D5 and 9B11 bind both human domain1-containing 4-1BB Fc(kih) molecules. The phage-derived clones 25G7binds human domain 2-containing hybrid 4-1BB Fc(kih) molecule. Thephage-derived clones 20G2 binds murine domain 1-containing hybrid 4-1BBFc(kih) molecule. A summary can be found in Table 58.

TABLE 58 Binding of hybrid 4-1BB Fc(kih) variants to anti-4-1BBantibodies 1 = binding; 0 = no binding. hybrid 4-1BB Fc(kih) Construct 1Construct 2 Construct 3 murine human human murine human 4-1BB 4-1BB4-1BB 4-1BB 4-1BB D1 Clone D1 (N-) D2 (C-) D1 (N-) D2 (C-) (N-) 12B3 0 11 25G7 1 0 0 11D5 0 1 1 9B11 0 1 1 20G2 1 0 0

Example 8 Functional Properties of Anti 4-1BB Binding Clones

8.1 Functional Properties of Anti-human 4-1BB Binding Clones

4-1BB serves as a co-stimulatory receptor and improves expansion,cytokine production and functional properties of T cells in aTCR-dependent manner. TCR-activation via surface immobilized anti-CD3antibody or peptide-pulsed antigen presenting cells inducesup-regulation of 4-1BB on T cells (Pollok et al. (1993) J. Immunol.150(3): 771-781) and support after engagement the immune response byboosting expansion and cytokine release (Hurtado et al. (1995) JImmunology 155(7), 3360-3367). To test boosting capacity of thegenerated anti-human 4-1BB antibodies, round-bottom suspension 96-wellplates (Greiner bio-one, cellstar, Cat.-No. 650185) were coated overnight with DPBS containing 2 μg/mL AffiniPure F(ab′)2 fragment goatanti-human IgG, Fcγ-fragment-specific (Jackson Immunoresearch, Cat.-No.109-006-008) and 2 μg/mL AffiniPure goat anti-mouse IgG,Fcγ-fragment-specific (Jackson Immunoresearch, Cat.-No 115-005-008).Plates were washed with DPBS to remove excessive molecules and wereblocked for 90 min at 37° C. with DPBS containing 1% (w/v) BSA(SIGMA-Aldrich, Cat-No. A3059-100G). Supernatant was removed and plateswere incubated with DPBS supplied with 1% (w/v) BSA and with or without10 ng/mL anti-human CD3 antibody (BioLegend, Cat.-No. 317315, cloneOKT3) for 90 min at 37° C. Plates were washed with DPBS and incubatedwith DPBS supplied with 1% (w/v) BSA and different concentrations oftitrated anti-human 4-1BB IgG1 P329 G LALA antibodies. Plates werewashed with DPBS and supernatant was aspirated.

Human PBMCs were isolated as described before (7.1.2) and activated inRPMI 1640 containing 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 2 ug/mL PHA-Land 200 U/mL Proleukin for 5 days. Cells were further cultured in RPMI1640 containing 10% (v/v) FBS, 1% (v/v) GlutaMAX-I and 200 U/mLProleukin for further 21 days at a density of 1-2×10⁶ cells/mL. Longtime cultured PBMCs were harvested washed and CD8 T cells were isolatedaccording to manufactures protocol using human CD8⁺ T cell isolation kit(Miltenyi Biotec, Cat.-No. 130-096-495). Preactivated and sorted CD8⁺ Tcells were seeded to 7×10⁴ cells/well in 200 μL/well T cell mediumconsisting of RPMI 1640 medium supplied with 10% FBS, 1% (v/v)GlutaMAX-I, 1 mM Sodium Pyruvate, 1% (v/v) MEM non-essential amino acidsand 50 μM β-Mercaptoethanol. Cells were incubated for 72 h, the last 4 hin the presence of Golgi-Stop (BD Bioscience, Cat.-No. 554724). Cellswere washed with DPBS and incubated for 30 min at 4° C. in 100 μL/wellDPBS containing 1:5000 diluted LIVE/DEAD Fixable Green Dead Cell Stain(Molecular Probes, Life Technologies, Cat.-No. L-23101). Afterwardscells were washed and incubated for 30 min at 4° C. in 50 μL/well FACSbuffer containing 0.5 μg/mL PerCP/Cy5.5-conjugated anti-human CD8 (mouseIgG1 κ, clone RPA-T8, BioLegend, Cat.-No. 301032), 0.5 μg/mLPE/Cy7-conjugated anti-human CD25 (mouse IgG1 κ, clone BC96, BioLegend,Cat.-No. 302612) and 1 μg/mL APC/Cy7-conjugated anti-human PD-1 (mouseIgG1 κ, clone EH12.2H7, BioLegend, Cat.-No. 329922). Cells were washedwith FACS buffer and resuspended in 50 μL/well in freshly preparedfixation/permeabilization solution (eBioscience, Cat.-No. 00-5523-00).After incubation for 30 min at 4° C., cells were washed with freshlyprepared permeabilization buffer (eBioscience, Cat.-No. 00-5523-00) andincubated for 1 h at 4° C. with 50 μL/well Perm-buffer containing 2μg/mL APC-labeled anti-human-IFNγ (mo IgG1 κ, clone B27, BD Pharmingen,Cat.-No. 554702) and 2 μg/mL PE-conjugated anti-human-TNFα(mo IgG1 κ,clone MAb11, BD Pharmingen, Cat.-No. 554513). Cells were washed andfixed with DPBS containing 1% formaldehyde. Cells were acquired the nextday using 2-laser Canto II (BD, DIVA software). Gates were set on CD8⁺ Tcells and frequency of TNFα and IFNγ secreting CD8⁺ T cells weredetermined. Using Graph Pad Prism (Graph Pad Software Inc.) data wasblotted and curves were calculated using non-linear regression curve fit(robust fit).

As described before in the absence of TCR- or CD3-stimulation 4-1BBengagement has no effect on CD8⁺ T cell function (Pollok 1995), whereasin the presence of suboptimal CD3-activation co-stimulation of 4-1BBincrease cytokine secretion (FIG. 31A or 31C). Sub-optimal activationvia CD3-antibody induces IFNγ-secretion in 30% of CD8⁺ T cells in thetotal CD8⁺ T cell population. Addition of 4-1BB-co-stimulation increasesthe IFNγ+ CD8 T cell population up to 55% in a concentration dependentmanner (FIG. 31B). Table 59 shows the corresponding EC₅₀ values. TNFαsecretion could be increased from 23% to 39% of total CD8+ T cellpopulation (FIG. 31D). Similar to their binding properties clones 12B3and 11D5 increase INFγ expression superior to 25G7 and 9B11 in frequencyand EC50. Therefore our generated clones are functional and can improveTCR-mediated T cell activation and function. If the anti-4-1BB-specificantibodies were not surface immobilized, they did not improve CD8⁺ Tcell activation (not shown).

TABLE 59 EC₅₀ values of increase of IFNγ secretion in activated CD8⁺ TCells Clone EC₅₀ CD8 [nM] 25G7 0.12 12B3 0.07 11D5 0.06 9B11 0.128.2 Functional Properties of Anti-mouse 4-1BB Binding Clone 20G2 InVitro

To prevent crosslinking of agonistic anti-4-1BB antibodies viaFc-receptors, mutation were introduced in the Fc-part of the antibodies.Similar to the P329G LALA mutation in human IgG1 antibodies a DAPGmutation was introduced into the Fc-part of mouse IgG1 molecules. Totest if this prevents the FcR-crosslinking and therefore activationcapacity, mouse splenocytes were activated with a sub-optimalconcentration of anti-mouse CD3 antibody and different concentration ofclone 20G2 as mouse IgG1 and mouse IgG1 DAPG.

Spleens were obtained from healthy female C57BL/6 mice (age 7-9 weeks,Charles River, France) and homogenized in 3 mL MACS buffer using C tubes(Miltenyi Biotec, Cat.-No. 130-096-334) and a gentle MACS OctoDissociator (Miltenyi Biotec, Cat.-No. 130-095-937). Aftercentrifugation (400×g, 8 min, RT) cell pellet was resuspended in 7 mlACK lysis buffer and erythrolysis was performed for 10 min at 37° C.Erythrolysis was stopped by addition of T cell medium consisting of RPMI1640 medium supplied with 10% FBS, 1% (v/v) GlutaMAX-I, 1 mM SodiumPyruvate, 1% (v/v) MEM non-essential amino acids and 50 μMβ-Mercaptoethanol. Splenocytes were washed with DPBS and filteredthrough a 70 μm cell strainer (Corning Cat.-No. 431751). Splenocyteswere resuspended to 2×10⁷ cells/mL in 37° C. DPBS and Cell ProliferationDye eFluor 670 (eBioscience, Cat.-No. 65-0840-90) were added to a finalconcentration of 2.5 mM. Cells were incubated for 10 min at 37° C.,labeling process was stopped by adding FBS and cells were washed. 200μL/well T cell medium containing 0.5 μg/mL anti-mouse CD3-specifichamster IgG (clone 145-2C11, BD Bioscience, Cat.-No. 553057), 0.01-50 nMtitrated anti-mouse 4-1BB-specific clone 20G2 mouse IgG1 or mouse IgG1DAPG or isotype control MOPC-21 mouse IgG1 (BD Bioscience, Cat.-No.554121) or isotype control DP47 moIgG1 DAPG and 1×10⁵ splenocytes weretransferred to wells of a 96-well round-bottom tissue culture plate(TPP, Cat.-No. 92097). Plates were incubated for 48 h. Cells werewashed, resuspended in 25 μL/well FACS buffer containing 1 μg/mLBV711-conjugated anti-mouse-CD8 rat IgG2a (clone 53-6.7, BioLegend,Cat.-No. 100748) and 2 μg/mL BV421-conjugated anti-mouse CD4 Rat IgG2a(clone RM4-5, BioLegend, Cat.-No. 100544) and incubated for 20 min at 4°C. Cells were washed and resuspended in 100 μL/well freshly preparedfixation/permeabilization solution (eBioscience, Cat.-No. 00-5523-00).After incubation for 30 min at 4° C. cells were washed with DPBS andresuspended in 100 μL/well freshly prepared permeabilization buffer(eBioscience, Cat.-No. 00-5523-00) containing 10 μg/mL Alexa Fluor488-conjugated anti-mouse Eomes rat IgG2a (clone DAN11MAG, eBioscience,Cat.-No. 53-4875-82) and 2 μg/mL PE-conjugated anti-mouse Granzyme B ratIgG2a (clone NGZB, eBioscience, Cat.-No. 12-8898-82). Cells wereincubated for 1 h at RT in the dark, washed with permeabilizationbuffer, resuspended in FACS buffer and acquired using the 5-laserFortessa (BD Bioscience, DIVA software). Gates were set on CD8⁺ T cellsand frequency of Eomes and Granzyme B expressing CD8⁺ T cells weredetermined. Using Graph Pad Prism (Graph Pad Software Inc.) data wasblotted and curves were calculated using non-linear regression curve fit(robust fit).

An optimal activation of CD8⁺ T cells induces Eomesodermin (Eomes)expression, a T-box transcription factor regulating the cytolyticeffector mechanisms of CD8⁺ T cells (Glimcher et al. 2004) and cytolyticenzymes like granzyme B. As shown in FIGS. 32A and 32B, the used 0.5μg/mL anti-mouse CD3 antibody concentration was sub-optimal and thefrequency of granzyme B expressing CD8⁺ T cells did not exceed above 30%and of eomesodermin (eomes) expressing CD8⁺ T cells did not exceed above18%. Only if a sufficient amount of anti-mouse 4-1BB clone 20G2 mouseIgG1 was added more CD8⁺ T cells started to express granzyme B (FIG.32A) and eomesodermin (FIG. 32B). This proofs, that clone 20G2 is anagonistic binder and is able to activated T cells by engaging mouse4-1BB. If crosslinking of clone 20G2 was impaired due to the DAPGmutation, no effect of improved activation was seen, which shows, thatalso with mouse cells crosslinking of the anti-4-1BB antibody is neededto induce 4-1BB down-stream signaling. Therefore the DAPG mutationprevents a sufficient crosslinking and presentation of the antibody viaFc-binding receptor. Non-mouse-4-1BB-specific isotype controls had noeffect on T cell activation.

8.3 Functional Properties of Anti-mouse 4-1BB Binding Clone 20G2 In Vivo

Treatment of mice with agonistic anti-mouse 4-1BB rat IgG2a leads to anaccumulation of activated T cells in the liver in tumor-free (Dubrot etal. (2010) Cancer Immunol Immunother. 59(8), 1223-33; Niu et al. (2007)J Immunol. 178(7):4194-213) and tumor-bearing mice (Wang et al. (2010) JImmunol. 185(12):7654-62; Kocak et al. (2006) Cancer Res.66(14):7276-84) and induce a hepatic inflammation. Similar a clinicalPhase II trial with anti-4-1BB antibody Urelumab (huIgG4) in stage IVmelanoma patients had to be terminated due to unusually high incidenceof grade 4 hepatitis (Ascierto P. et al. (2010) Semin Oncol. 37(5),508-16). To test, if anti-mouse 4-1BB mouse IgG1 can induce a similar Tcell accumulation into the liver as reported agonistic anti-mouse 4-1BBrat IgG2a antibodies and further, if the DAPG mutation can prevent this,naïve healthy female C57BL/6 mice (age 7-9 weeks, Charles River, France)were treated with anti-mouse 4-1BB IgG1 and IgG DAPG clone 20G2. Micewere kept and house as described herein before. 150 μg antibody wereinjected intravenously into the tail vain weekly for 3 weeks. Mice weresacrificed by cervical dislocation one day after second injection andone day and eight days after third injection. Spleens and liver weredissected and stored on ice in RPMI 1640 supplemented with 10% (v/v)heat-inactivated FBS and 1% (v/v) GlutaMAX-I. Splenocytes were isolatedas described before (8.2). Leucocytes from the liver were isolated asfollowing: each liver was transferred to C tube (Miltenyi Biotec,Cat.-No. 130-096-334) containing 5 mL RPMI 1640 supplied with 0.3 mg/mLDispase II (Roche Applied Science, Cat.-No.: 04942078001), 1 μg/mL DnaseI (Roche Applied Science, Cat.-No.: 11284932001) and 2 mg/mL CollagenaseD (Roche Applied Science, Cat.-No.: 11 088 882001), tissue was disruptedusing gentle MACS Octo Dissociator using the program “m_liver_01”(Miltenyi Biotec, Cat.-No. 130-095-937) and livers were incubated for 30min at 37° C. The digested livers were disrupted a second time using theMACS Octo Dissociator using the program “m_liver_02”. Aftercentrifugation (400×g, 8 min, RT) cell pellet was resuspended in 7 mlACK lysis buffer and erythrolysis was performed for 10 min at 37° C.Erythrolysis was stopped by addition of RPMI 1640 medium supplied with10% FBS and 1% (v/v) GlutaMAX-I. 10⁶ cells/well splenocytes orleucocytes from the liver were transferred to a round-bottom 96-wellsuspension cell plate (Greiner bio-one, cellstar, Cat.-No. 650185) andwashed once with DPBS. Cells were resuspended in 100 μL/well containing1:5000 diluted LIVE/DEAD Fixable Green Dead Cell Stain Kit (LiveTechnologies Cat.-No. L-23101) and incubated for 30 min at 4° C.Afterwards cells were washed and resuspended in 50 μL/well inFACS-buffer containing 6 μg/mL PE-conjugated anti-mouse CD137 syrianhamster IgG (clone 17B5, BioLegend, Cat.-No. 106106) or Syrian hamsterisotype control (clone SHG-1, BioLegend, Cat.-No. 402008), 2 μg/mLPerCP/Cy5.5-conjugated anti-mouse TCR-β Armenian hamster IgG (cloneH57-597, BioLegend, Cat.-No. 109228), 5 μg/mL PE/Cy7-conjugatedanti-mouse CD25 rat IgG1 (clone PC61, BD Bioscience, Cat.-No. 552880) orrat IgG1 γ isotype control (clone A110-1, BD Bioscience, Cat.-No.552869), 2 μg/mL Alexa Fluor 700-labeled anti-mouse CD8a ratIgG2a κ(clone 53-6.7, BD Bioscience, Cat.-No. 557959), 0.25 μg/mLBV421-conjugated anti-mouse CD4 rat igG2b κ (clone GK1.5, BioLegend,Cat.-No. 100438), 4 μg/mL BV605-conjugated anti-mouse-CD11c Armenianhamster IgG (clone N418, BioLegend, Cat.-No. 117333) or Armenian hamsterisotype control (clone HTK888, BioLegend, Cat.-No. 400943) and incubatedfor 30 min at 4° C. Cells were washed and resuspended in 100 μL/wellfreshly prepared fixation/permeabilization solution (eBioscience,Cat.-No. 00-5523-00). After incubation for 30 min at 4° C. cells werewashed with freshly prepared permeabilization buffer (eBioscience,Cat.-No. 00-5523-00) and resuspended in 50 μL/well permeabilizationbuffer containing 0.2 μg/mL eF660-conjugated anti-mouse Ki67 rat IgG2a κ(clone SolA15, eBioscience, Cat.-No. 50-5698-82). Cells were incubatedfor 30 min at 4° C., washed twice with permeabilization buffer and fixedwith 50 μL/well DPBS containing 1% formaldehyde. The next day cells wereacquired using the 5-laser Fortessa (BD Bioscience, DIVA software). Datawas analyzed using FlowJo (FlowJo, LLC), gates were set on CD8⁺ or CD4⁺T cells and frequency of CD137⁺, CD25⁺, CD11c⁺ and Ki67⁺ T cells weredetermined. Using Graph Pad Prism (Graph Pad Software Inc.) data wasblotted.

As shown in FIG. 33, only treatment of mice with anti-mouse 4-1BB clone20G2 mouse IgG1 induce the accumulation, proliferation and increased4-1BB (CD137) expression on CD8⁺ T cells in the liver, whereaspreventing crosslinking via FcR-binding with the DAPG mutation bypassesactivation of CD8⁺ T cells. Preventing FcR-crosslinking with the DAPGmutation prevents the activation of CD8⁺ T cells in the liver (FIG. 33),although both molecules show similar binding properties to mouse CD137expressed on T cells (FIGS. 25B and 25D) and therefore feature the samepotential activation properties.

Example 9 Preparation, Purification and Characterization of BispecificBivalent Antibodies Targeting 4-1BB and a Tumor Associated Antigen (TAA)

9.1 Generation of Bispecific Bivalent Antibodies Targeting 4-1BB andFibroblast Activation Protein (FAP) (2+2 Format)

Bispecific agonistic 4-1BB antibodies with bivalent binding for 4-1BBand for FAP were prepared. The crossmab technology was applied to reducethe formation of wrongly paired light chains as described inInternational patent application No. WO 2010/145792 A1.

The generation and preparation of the FAP binders is described in WO2012/020006 A2, which is incorporated herein by reference.

In this example, a crossed Fab unit (VHCL) of the FAP binder 28H1 wasC-terminally fused to the heavy chain of an anti-4-1BB hu IgG1 using a(G4S)₄ connector sequence. This heavy chain fusion was co-expressed withthe light chain of the anti-4-1BB and the corresponding FAP crossedlight chain (VLCH1). The Pro329Gly, Leu234Ala and Leu235Ala mutationshave been introduced in the constant region of the heavy chains toabrogate binding to Fc gamma receptors according to the method describedin International Patent Appl. Publ. No. WO 2012/130831 A1. The resultingbispecific, bivalent construct is analogous to the one depicted in FIG.34A.

Table 60 shows, respectively, the nucleotide and amino acid sequences ofmature bispecific, bivalent anti-4-1BB/anti-FAP human IgG1 P329GLALAantibodies.

TABLE 60 Sequences of bispecific, bivalent anti-4-1BB/anti-FAP humanIgG1 P329GLALA antigen binding molecules SEQ ID NO: Description Sequence286 (12B3) VHCH1- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCA AGTCCTTCAACCGGGGCGAGTGC 259VLCL-Light see Table 40 chain 1 (12B3) (nucleotide sequence) 215VLCH1-Light GAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCTGA chain 2 (28H1)GCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCC (nucleotideAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGA sequence)AGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGAGCTCCGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCCAGCAGCAAGAGCACCTCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCC AAGAGCTGCGAC 287 (12B3) VHCH1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 261 VLCL-Light see Table 40chain 1 (12B3) 217 VLCH1-LightEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPG chain 2 (28H1)QAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CD 288 (25G7) VHCH1-GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG Heavy chain-CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT (28H1) VHCLTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGC (nucleotideTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGG sequence)TAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAAC CGGGGCGAGTGC 263 VLCL-Lightsee Table 40 chain 1 (25G7) (nucleotide sequence) 215 VLCH1-Lightsee above chain 2 (28H1) (nucleotide sequence) 289 (25G7) VHCH1-EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP Heavy chain-GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ (28H1) VHCLMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 265 VLCL-Light see Table 40chain 1 (25G7) 217 VLCH1-Light see above chain 2 (28H1) 290(11D5) VHCH1- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAA CCGGGGCGAGTGC 267 VLCL-Lightsee Table 40 chain 1 (11D5) (nucleotide sequence) 215 VLCH1-Lightsee above chain 2 (28H1) (nucleotide sequence) 291 (11D5) VHCH1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 269 VLCL-Light see Table 40chain 1 (11D5) 217 VLCH1-Light see above chain 2 (28H1) 292(9B11) VHCH1- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG Heavy chain-CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG (28H1) VHCLGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGG (nucleotideCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCC sequence)CTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGAGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCTAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGGGGAGGCGGAGGATCTGGCGGAGGCGGATCCGGTGGTGGCGGATCTGGGGGCGGTGGATCTGAGGTGCAGCTGCTGGAATCTGGGGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGGCTGTCCTGCGCTGCTTCCGGCTTTACCTTCTCCAGCCACGCCATGAGTTGGGTGCGCCAGGCACCCGGAAAAGGACTGGAATGGGTGTCAGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGATAGCGTGAAGGGCCGGTTCACCATCTCTCGGGATAACAGCAAGAATACTCTGTACCTGCAGATGAACTCCCTGCGCGCTGAAGATACCGCTGTGTATTACTGCGCCAAGGGCTGGCTGGGCAACTTCGATTACTGGGGCCAGGGAACCCTCGTGACTGTCTCGAGCGCTTCTGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACTGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCAGCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGT CCTTCAACCGGGGCGAGTGC 271VLCL-Light see Table 40 chain 1 (9B11) (nucleotide sequence) 215VLCH1-Light see above chain 2 (9B11) (nucleotide sequence) 293(9B11) VHCH1- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP Heavy chain-GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYME (28H1) VHCLLSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 273 VLCL-Light see Table 40chain 1 (9B11) 217 VLCH1-Light see above chain 2 (9B11)

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

The bispecific anti-4-1BB/anti-FAP constructs were produced byco-transfecting HEK293-EBNA cells with the mammalian expression vectorsusing polyethylenimine. The cells were transfected with thecorresponding expression vectors in a 1:1:1 ratio (“vector heavychain”:“vector light chain1”:“vector light chain2”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes by 210×g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO₂ atmosphere. After theincubation, 160 mL F17 medium was added and cells were cultured for 24hours. One day after transfection 1 mM valproic acid and 7% Feed wereadded. After culturing for 7 days, the cell supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of bispecific constructs from cell culture supernatants wascarried out by affinity chromatography using Protein A as describedabove for purification of antigen-Fc fusions and antibodies.

The protein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl solution of pH 6.0.

The protein concentration of purified bispecific constructs wasdetermined by measuring the OD at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of the bispecific constructs were analyzed byCE-SDS in the presence and absence of a reducing agent (Invitrogen, USA)using a LabChipGXII (Caliper). The aggregate content of bispecificconstructs was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in a 25 mM K₂HPO₄, 125 mMNaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN₃, pH 6.7running buffer at 25° C. (Table 61).

TABLE 61 Biochemical analysis of bispecific, bivalentanti-4-1BB/anti-FAP IgG1 P329G LALA antigen binding molecules MonomerYield Clone [%] [mg/l] 12B3/FAP P329GLALA 97.6 6.8 IgG1 2 + 2 25G7/FAPP329GLALA 98.4 13 IgG1 2 + 2 11D5/FAP P329GLALA 100 8.7 IgG1 2 + 29B11/FAP P329GLALA 99 0.3 IgG1 2 + 29.2 Binding of Bispecific Bivalent Antibodies Targeting 4-1BB and FAP9.2.1 Surface Plasmon Resonance (Simultaneous Binding)

The capacity of binding simultaneously human 4-1BB Fc(kih) and human FAPwas assessed by surface plasmon resonance (SPR). All SPR experimentswere performed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany).

Biotinylated human 4-1BB Fc(kih) was directly coupled to a flow cell ofa streptavidin (SA) sensor chip. Immobilization levels up to 1000resonance units (RU) were used. The bispecific antibodies targeting4-1BB and FAP were passed at a concentration range of 250 nM with a flowof 30 μL/minute through the flow cells over 90 seconds and dissociationwas set to zero sec. Human FAP was injected as second analyte with aflow of 30 μL/minute through the flow cells over 90 seconds at aconcentration of 250 nM (FIG. 34B). The dissociation was monitored for120 sec. Bulk refractive index differences were corrected for bysubtracting the response obtained in a reference flow cell, where noprotein was immobilized. All bispecific constructs could bindsimultaneously human 4-1BB and human FAP (FIGS. 35A-35D).

9.2.2 Binding on Cells

9.2.2.1 Binding on Human 4-1BB Expressing Cells: Resting and ActivatedHuman Peripheral Mononuclear Blood Leukocytes (PBMC)

Human PBMCs were isolated, activated to induce 4-1BB and used to testbinding properties of antibodies to human 4-1BB as already describedunder Example 7.1.2.

To show that Fc-fused FAP-targeting does not influence the binding ofthe anti-4-1BB specific clones or the detection via PE-conjugated humanIgG Fc-specific goat IgG F(ab′), we tested the binding capacity ofFAP-targeted or DP47-untargeted 2+2 constructs compared to the parentalhuIgG P329G LALA formats to resting human CD4⁺ and CD8⁺ T cells (FIGS.38A-38F) and activated human CD4⁺ and CD8⁺ T cells (FIGS. 39A-39F).Similar to FIG. 23 also the FAP-targeted or DP47-targeted 2+2 human4-1BB-specific molecules did not bind to resting T cells (FIGS. 38A-38F)but mainly to activated CD8⁺ T cells (FIGS. 39 D, 39E and 39F). Theconversion into the FAP-targeting or DP47-non-targeting 2+2 formats didnot change the binding behaviour to 4-1BB-expressing T cellsdramatically. Differences may be explained by the detection withFc-specific polyclonal secondary antibody (e.g. Fc-fusion may maskepitopes and reduce the detection). EC₅₀ values varied from donor todonor depending on the amount of expressed 4-1BB after the activation.This explains the difference of EC₅₀ values listed in Table 62 comparedwith those of the parental antibodies as shown in Table 48. Area underthe curve of binding to activated CD8⁺ T cells are shown in FIG. 40.

TABLE 62 EC₅₀ values of binding to activated human CD8 T cells CloneEC₅₀ [nM] 25G7 11.14 25G7/FAP 28H1 2 + 2 13.21 25G7/DP47 2 + 2 50.0312B3 0.43 12B3/FAP 28H1 2 + 2 1.99 11D5 1.35 11D5/FAP 28H1 2 + 2 7.919.2.2.2 Binding to Human FAP-expressing Tumor Cells

For binding assays on cell-surface expressed Fibroblast ActivationProtein (FAP) NIH/3T3-huFAP clone 19 cell line or human melanoma cellline WM-266-4 (ATCC CRL-1676) were used. NIH/3T3-huFAP clone 19 wasgenerated by transfection of mouse embryonic fibroblast NIH/3T3 cells(ATCC CRL-1658) with the expression pETR4921 plasmid encoding human FAPunder a CMV promoter. Cells were maintained in the presence of 1.5 μg/mLpuromycin (InvivoGen, Cat.-No.: ant-pr-5). 2×10⁵ of FAP expressing tumorcells were added to each well of a round-bottom suspension cell 96-wellplates (Greiner bio-one, cellstar, Cat.-No. 650185). Cells were washedonce with 200 μL DPBS, resuspended in 100 μL/well of 4° C. cold DPBSbuffer containing 1:5000 diluted Fixable Viability Dye eFluor 450(eBioscience, Cat. No. 65 0863 18) or Fixable Viability Dye eFluor 660(eBioscience, Cat.-No. 65-0864-18) and incubated for 30 minutes at 4° C.Cells were washed once with 200 μL 4° C. cold DPBS buffer, resuspendedin 50 μL/well of 4° C. cold FACS buffer containing different titratedconcentrations of 4-1BB-specific FAP-targeted and non-targetedantibodies followed by an incubation for 1 hour at 4° C. After washingfour times with 200 μL/well, cells were stained with 50 μL/well of 4° C.cold FACS buffer containing 2.5 μg/mL PE-conjugated AffiniPureanti-human IgG Fcγ-fragment-specific goat F(ab′)2 fragment (JacksonImmunoResearch, Cat.-No. 109-116-098) or 30 μg/mL FITC-conjugatedAffiniPure anti-human IgG Fcγ-fragment-specific goat F(ab′)2 fragment(Jackson ImmunoResearch, Cat. No. 109 096 098) for 30 minutes at 4° C.Cells were washed twice with 200 μL 4° C. FACS buffer and cells wereresuspended in 50 μL/well DPBS containing 1% formaldehyde for fixation.The same or next day cells were resuspended in 100 μL FACS-buffer andacquired using 5-laser LSR-Fortessa (BD Bioscience with DIVA software)or MACSQuant Analyzer 10 (Miltenyi Biotec).

As shown in FIGS. 41A-41F, all tested 2+2 FAP-targeted molecules, butnot the DP47-targeted or parental huIgG1 P293G LALA formats containingthe 4-1BB-binding clone 25G7, 11D5 and 12B3 clones bind efficiently andto human FAP-expressing cells. Therefore the tested FAP (28H1)-targeted2+2 constructs can specifically bind to FAP-expressing cells. In FIG. 42a summary as area under (AUC) the curve of NIH/3T3-huFAP binding curvesis shown. Tested formats and FAP-binding clones are indicated below thegraph as pictogram. It shows that all tested FAP-targeted 2+2 formatsshow a similar AUC.

TABLE 63 EC₅₀ values of binding to FAP expressing cell lineNIH/3T3-huFAP clone 19 and WM-266-4 EC₅₀ [nM] with NIH/3T3-huFAP cloneEC₅₀ [nM] with Clone 19 cells WM-266-4 12B3/FAP 28H1 2 + 2 12 n.d.25G7/FAP 28H1 2 + 2 0.4 n.d. 11D5/FAP 28H1 2 + 2 0.9 0.7 25G7/DP47 2 + 20.4 n.d.9.2.3 NFκB Activation9.2.3.1 Generation of HeLa Cells Expressing Human 4-1BB andNF-κB-luciferase

The cervix carcinoma cell line HeLa (ATCC CCL-2) was transduced with aplasmid based on the expression vector pETR10829, which contains thesequence of human 4-1BB (Uniprot accession Q07011) under control of aCMV-promoter and a puromycin resistance gene. Cells were cultured inDMEM medium supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I and 3μg/mL puromycin.

4-1BB-transduced HeLa cells were tested for 4-1BB expression by flowcytometry: 0.2×10⁶ living cells were resuspended in 100 μL FACS buffercontaining 0.1 μg PerCP/Cy5.5 conjugated anti-human 4-1BB mouse IgG1κclone 4B4-1 (BioLegend Cat. No. 309814) or its isotype control(PerCP/Cy5.5 conjugated mouse IgG1κ isotype control antibody clone MOPC21, BioLegend Cat. No. 400150) and incubated for 30 minutes at 4° C.Cells were washed twice with FACS buffer, resuspended in 300 μL FACSbuffer containing 0.06 μg DAPI (Santa Cruz Biotec, Cat. No. Sc-3598) andacquired using a 5-laser LSR-Fortessa (BD Bioscience, DIVA software).Limited dilutions were performed to generate single clones as described:human-4-1BB-transduced HeLa cells were resuspended in medium to adensity of 10, 5 and 2.5 cells/ml and 200 μl of cell suspensions weretransferred to round bottom tissue-culture treated 96-well plates (6plates/cell concentration, TPP Cat. No. 92697). Single clones wereharvested, expanded and tested for 4-1BB expression as described above.The clone with the highest expression of 4-1BB (clone 5) was chosen forsubsequent transfection with the NF-κB-luciferase expression-vector5495p Tranlucent HygB. The vector confers transfected cells both withresistance to Hygromycin B and capacity to express luciferase undercontrol of NF-kB-response element (Panomics, Cat. No. LR0051). Fortransfection human-4-1BB HeLa clone 5 cells were cultured to 70%confluence. 50 μg (40 μL) linearized (restriction enzymes AseI and SalI)5495p Tranlucent HygB expression vector were added to a sterile 0.4 cmGene Pulser/MicroPulser Cuvette (Biorad, Cat.-No, 165-2081). 2.5×10⁶human-4-1BB HeLa clone 5 cells in 400 μl supplement-free DMEM mediumwere added and mixed carefully with the plasmid solution. Transfectionof cells was performed using a Gene Pulser Xcell total system (Biorad,Cat No. 165 2660) under the following settings: exponential pulse,capacitance 500 μF, voltage 160 V, resistance ∞. Immediately after thepulse transfected cells were transferred to a 75 cm² cm tissue cultureflask (TPP, Cat. No. 90075) with 15 mL 37° C. warm DMEM medium suppliedwith 10% (v/v) FBS and 1% (v/v) GlutaMAX I. On the next day, culturemedium containing 3 μg/mL Puromycin and 200 μg/mL Hygromycin B (Roche,Cat. No. 10843555001) was added. Surviving cells were expanded andlimited dilution was performed as described above to generate singleclones.

Clones were tested for 4-1BB expression as described above and forNF-κB-Luciferase activity as following: Clones were harvested inselection medium and counted using a Cell Counter Vi-cell xr 2.03(Beckman Coulter, Cat. No. 731050). Cells were set to a cell density of0.33×10⁶ cells/mL and 150 μL of this cell suspension were transferred toeach well of a sterile white 96-well flat bottom tissue culture platewith lid (greiner bio one, Cat. No. 655083). Cells were incubated at 37°C. and 5% CO₂ overnight in a cell incubator (Hera Cell). The next day 50μL of medium containing different concentrations of recombinant humantumor necrosis factor alpha (rhTNFα, PeproTech, Cat.-No. 300 01A) wereadded to each well of a 96-well plate resulting in final concentrationof rhTNFα of 100, 50, 25, 12.5, 6.25 and 0 ng/well. Cells were incubatedfor 6 hours at 37° C. and 5% CO₂ and then washed three times with 200μL/well DPBS. Reporter Lysis Buffer (Promega, Cat-No: E3971) was addedto each well (40 μl) and the plates were stored over night at −20° C.The next day frozen cell and Detection Buffer (Luciferase 1000 AssaySystem, Promega, Cat. No. E4550) were thawed to room temperature. 100 uLof detection buffer were added to each well and the plate was measuredas fast as possible using a SpectraMax M5/M5e microplate reader and theSoftMax Pro Software (Molecular Devices). Measured units of releasedlight for 500 ms/well (URLs) above control (no rhTNFα added) were takenas luciferase activity. The HeLa-hu4-1BB-NF-κB-luc clone 26 exhibitingthe highest luciferase activity and a considerable level of4-1BB-expression and was chosen for further use.

9.2.3.2 NFκB Activation in HeLa Cells Expressing Human 4-1BB ReporterCells Co-cultured with FAP-expressing Tumor Cells

HeLa-hu4-1BB-NF-κB-luc clone 26 cells were harvested and resuspended inDMEM medium supplied with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I to aconcentration of 0.2×10⁶ cells/ml. 100 μl (2×10⁴ cells) of this cellsuspension were transferred to each well of a sterile white 96-well flatbottom tissue culture plate with lid (greiner bio one, Cat. No. 655083)and the plate were incubated at 37° C. and 5% CO₂ overnight. The nextday 50 μL of medium containing titrated FAP-targeted anti-human 4-1BBconstructs or their parental huIgG1 P329G LALA antibodies were added.FAP-expressing NIH/3T3-huFAP clone 19 or WM-266-4 were resuspended inDMEM medium supplied with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I to aconcentration of 2×10⁶ cells/ml.

Suspension of FAP-expressing tumor cell (50 μl) or medium as negativecontrol (no FAP⁺ tumor cells) were added to each well and plates wereincubated for 6 hours at 37° C. and 5% CO₂ in the cell incubator. Cellswere washed twice with 200 μL/well DPBS. 40 μl freshly prepared ReporterLysis Buffer (Promega, Cat-No: E3971) were added to each well and theplate were stored over night at −20° C. The next day frozen cell plateand Detection Buffer (Luciferase 1000 Assay System, Promega, Cat. No.E4550) were thawed at room temperature. 100 μL of detection buffer wereadded to each well and luciferase activity was measured as fast aspossible using a SpectraMax M5/M5e microplate reader and a SoftMax ProSoftware (Molecular Devices).

FAP (28H1)-targeted clone 25G7, 11D5 or 12B3 2+2 constructs triggeredactivation of the NFκB signaling pathway in the reporter cell line inthe presence of NIH/3T3-huFAP cells (FIGS. 43C, 43F and 43I) in aconcentration-dependend manner. In contrast, the untargeted controlmolecule (4-1BB (25G7)×DP47 2+2) or the parental huIgG1 P329G LALAantibodies failed to trigger such an effect at any of the testedconcentrations. This activity of tested FAP-targeted anti-human 4-1BB2+2 antibodies was strictly dependent on a high expression of FAP at thecell surface of added crosslinking cells as no NF-κB activation could bedetected in the absence FAP-expressing tumor cells (FIGS. 43 A, 43D and43G) or in the presence of a tumor cell line expressing less FAP(WM-266-4) (FIGS. 43B, 43E and 43H). Therefore the activation depends onthe FAP-expression strength and not on affinity strength of theanti-4-1BB binder because the activation potency between 25G7×FAP 2+2,11D5×FAP 2+2 and 12B3×FAP 2+2 (FIGS. 44A and 44B and Table 64) do notcorrelate with their 4-1BB binding affinity (FIG. 42).

TABLE 64 EC₅₀ values of activation of the NFκB-controlled Luciferase in4-1BB expressing reporter cell lines in the presence of FAP-expressingNIH/3T3-huFAP clone 19 cells EC₅₀ [nM] with NIH/3T3- Clone huFAP clone19 12B3/FAP 28H1 2 + 2 0.2 11D5/FAP 28H1 2 + 2 0.2 25G7/FAP 28H1 2 + 20.1

Example 10 Preparation, Purification and Characterization of BispecificMonovalent Antibodies Targeting 4-1BB and a Tumor Associated Antigen(TAA)

10.1 Generation of Bispecific Antibodies Targeting 4-1BB and FibroblastActivation Protein (FAP) in Monovalent Format (1+1 Format)

Bispecific agonistic 4-1BB antibodies with monovalent binding for 4-1BBand for FAP were prepared. The crossmab technology was applied to reducethe formation of wrongly paired light chains as described inInternational patent application No. WO 2010/145792 A1.

The generation and preparation of the FAP binders is described in WO2012/020006 A2, which is incorporated herein by reference.

The bispecific construct binds monovalently to 4-1BB and to FAP (FIG.34C). It contains a crossed Fab unit (VHCL) of the FAP binder fused tothe knob heavy chain of an anti-4-1BB huIgG1 (containing the S354C/T366Wmutations). The Fc hole heavy chain (containing theY349C/T366S/L368A/Y407V mutations) is fused to a Fab against anti-4-1BB.Combination of the targeted anti-FAP-Fc knob with the anti-4-1BB-Fc holechain allows generation of a heterodimer, which includes a Fab thatspecifically binds to FAP and a Fab that specifically binds to 4-1BB.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fc gamma receptors according to the method described inInternational Patent Appl. Publ. No. WO 2012/130831 A1.

The bispecific monovalent anti-4-1BB and anti-FAP huIgG1 P329GLALA wereproduced by co-transfecting HEK293-EBNA cells with the mammalianexpression vectors using polyethylenimine. The cells were transfectedwith the corresponding expression vectors in a 1:1:1:1 ratio (“vectorknob heavy chain”:“vector light chain1”:“vector hole heavychain”:“vector light chain2”).

The resulting bispecific, monovalent constructs were produced andpurified as described for the bispecific bivalent anti-4-1BB andanti-FAP huIgG1 P329GLALA (see Example 9.1). The nucleotide and aminoacid sequences can be found in Table 65.

TABLE 65 cDNA and amino acid sequences of mature bispecific monovalentanti-4-1BB/anti-FAP huIgG1 P329GLALA kih antibodies SEQ ID NO:Description Sequence 228 (28H1) VHCL-heavyGAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGC chain holeAGCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCC (nucleotide sequence)GGCTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCTAGCGTGGCCGCTCCCAGCGTGTTCATCTTCCCACCCAGCGACGAGCAGCTGAAGTCCGGCACAGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 215 (28H1) VLCH1-LightGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAGCCT chain 2 (nucleotideGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAGCC sequence)AGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCATCGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAAATCAAGAGCTCCGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCCAGCAGCAAGAGCACCTCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCT GCGAC 229 (28H1) VHCL-heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQ chain holeAPGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 217 (28H1)VLCH1-Light EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQK chain 2PGQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCD 294 (12B3)VHCH1-heavy CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 259 (12B3) VLCL-Light see Table 40chain 1 (nucleotide sequence) 295 (12B3) VHCH1-heavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 261 (12B3)VLCL-Light see Table 40 chain 1 296 (25G7) VHCH1-heavyGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC chain knobAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC (nucleotide sequence)GGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAA 263(25G7) VLCL-Light see Table 40 chain 1 (nucleotide sequence) 297 (25G7)VHCH1-heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ chain knobAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 265 (25G7)VLCL-Light see Table 40 chain 1 298 (11D5) VHCH1-heavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGAC CACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCTCCGGGTAAA 267 (11D5)VLCL-Light see Table 40 chain 1 (nucleotide sequence) 299 (11D5)VHCH1-heavy QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 269 (11D5)VLCL-Light see Table 40 chain 1 300 (9B11) VHCH1-heavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA chain knobAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCTAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACAAGTGGAGGAACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACCGTGCCCTCTAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCTTGCCCTGCCCCTGAAGCTGCTGGTGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 271 (9B11) VLCL-Light see Table 40chain 1 (nucleotide sequence) 301 (9B11) VHCH1-heavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ chain knobAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 273 (9B11)VLCL-Light see Table 40 chain 1

All genes were transiently expressed under control of a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells.

The bispecific anti-4-1BB/anti-FAP constructs were produced byco-transfecting HEK293-EBNA cells with the mammalian expression vectorsusing polyethylenimine. The cells were transfected with thecorresponding expression vectors in a 1:1:1:1 ratio (“vector heavy knobchain”:“vector heavy hole chain”:“vector light chain1”:“vector lightchain2”).

For production in 500 mL shake flasks, 400 million HEK293 EBNA cellswere seeded 24 hours before transfection. For transfection cells werecentrifuged for 5 minutes by 210×g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were mixed in 20 mL CD CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI,the solution was vortexed for 15 seconds and incubated for 10 minutes atroom temperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3 hoursat 37° C. in an incubator with a 5% CO₂ atmosphere. After theincubation, 160 mL F17 medium was added and cells were cultured for 24hours. One day after transfection 1 mM valproic acid and 7% Feed wereadded. After culturing for 7 days, the cell supernatant was collected bycentrifugation for 15 minutes at 210×g. The solution was sterilefiltered (0.22 μm filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4° C.

Purification of bispecific constructs from cell culture supernatants wascarried out by affinity chromatography using Protein A as describedabove for purification of antigen/Fc fusion molecules or antibodies. Theprotein concentration of purified bispecific constructs was determinedby measuring the OD at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the bispecific constructs were analyzed by CE-SDS in thepresence and absence of a reducing agent (Invitrogen, USA) using aLabChipGXII (Caliper). The aggregate content of bispecific constructswas analyzed using a TSKgel G3000 SW XL analytical size-exclusion column(Tosoh) equilibrated in a 25 mM K2HPO4, 125 mM NaCl, 200 mM L-ArginineMonohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.

TABLE 66 Biochemical analysis of bispecific, monovalentanti-4-1BB/anti-FAP IgG1 P329G LALA antigen binding molecules YieldMonomer Clone [mg/l] [%] 12B3/FAP P329GLALA 14 94.8 IgG1 1 + 1 25G7/FAPP329GLALA 33 92.2 IgG1 1 + 110.2 Preparation, Purification and Characterization of FAP Antigens asScreening Tools

In order to test the binding to FAP, DNA sequences encoding theectodomains of human, mouse or cynomolgus FAP fused to a C-terminalHisTag were cloned in an expression vector containing a chimeric MPSVpromoter consisting of the MPSV core promoter combined with the CMVpromoter enhancer fragment. The expression vector also contains the oriPregion for episomal replication in EBNA (Epstein Barr Virus NuclearAntigen) containing host cells. The amino acid and nucleotide sequencesof a His-tagged human FAP ECD is shown in SEQ ID NOs 85 and 86,respectively. SEQ ID NOs 88 and 89 show the amino acid and nucleotidesequences, respectively, of a His-tagged mouse FAP ECD. SEQ ID NOs 90and 91 show the amino acid and nucleotide sequences, respectively, of aHis-tagged cynomolgus FAP ECD.

The FAP antigens were produced by co-transfecting HEK293-EBNA cells withthe mammalian expression vectors using polyethylenimine (PEI;Polysciences Inc.).

For a 200 mL production in 500 mL shake flasks, 300 million HEK293 EBNAcells were seeded 24 hours before transfection in 100% F17+6 mMGlutamine. For transfection, 400 million cells were centrifuged for 5minutes at 210×g, and supernatant was replaced by 20 mL pre-warmedCD-CHO medium (Gibco). Expression vectors were mixed in 20 mL CD-CHOmedium to a final amount of 200 μg DNA. After addition of 540 μL PEI (1mg/mL) (Polysciences Inc.), the solution was vortexed for 15 seconds andincubated for 10 minutes at room temperature. Afterwards, resuspendedcells were mixed with the DNA/PEI solution, transferred to a 500 mLshake flask and incubated for 3 hours at 37° C. in an incubator with a5% CO2 atmosphere and shaking at 165 rpm. After the incubation, 160 mLF17 medium and supplements (1 mM valproic acid, 5 g/l Pepsoy and 6 mML-Glutamine) were added and cells cultivated for 24 hours. 24 h aftertransfection the cells were then supplement with an amino acid andglucose feed at 12% final volume (24 mL). After cultivation for 7 days,the cell supernatant was collected by centrifugation for 45 minutes at2000-3000×g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Purification of the antigens from cell culture supernatants was carriedout in a two step purification with first an affinity chromatographystep using either a 5 ml IMAC column (Roche) or a 5 ml NiNTA column(Qiagen) followed by a size exclusion chromatography using a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine,140 mM NaCl, pH6.0 or 2 mM MOPS 150 mM NaCl 0.02% NaN3 pH 7.3,respectively.

For affinity chromatography, using the IMAC column (Roche),equilibration was performed with 25 mM Tris-HCl, 500 mM sodium Chloride,20 mM imidazole, pH8.0 for 8 CVs. The supernatant was loaded and unboundprotein washed out by washing with 10 CVs of 25 mM Tris-HCl, 500 mMsodium Chloride, 20 mM imidazole, pH8.0. The bound protein was elutedusing a linear gradient of 20 CVs (from 0-100%) of 25 mM Tris-HCl, 500mM sodium Chloride, 500 mM imidazole, pH8.0 followed by a step at 100%for 8 CVs.

For the affinity chromatography using the NiNTA column (Qiagen),equilibration was performed with 50 mM Sodium Phosphate, 300 mM SodiumChloride, pH8.0 for 8 CVs. The supernatant was loaded and unboundprotein was removed by washing with 10 column volumes of 50 mM SodiumPhosphate, 300 mM Sodium Chloride, pH8.0. The bound protein was elutedusing a linear gradient of 20 CVs (from 0 to 100%) of 50 mM SodiumPhosphate, 300 mM Sodium Chloride, 500 mM imidazole, pH7.4 followed by astep of 5CVs of 50 mM Sodium Phosphate, 300 mM Sodium Chloride, 500 mMimidazole, pH7.4. The column was then re-equilibrated with 8 CVs of 50mM Sodium Phosphate, 300 mM Sodium Chloride, pH8.0.

The collected fractions were then supplemented with 1/10 (v/v) of 0.5 MEDTA, pH8.0. The protein was concentrated in Vivaspin columns (30 kD cutoff, Sartorius) and filtered prior to loading on a HiLoad Superdex 200column (GE Healthcare) equilibrated with 2 mM MOPS 150 mM NaCl 0.02%NaN3 pH 7.3 or 20 mM Histidine, 140 mM NaCl, pH6.0.

The protein concentration of purified antigens was determined bymeasuring the OD at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the antigens were analyzed by CE-SDS in the presence andabsence of a reducing agent (Invitrogen) using a LabChipGXII (Caliper).The aggregate content of the antigens was analyzed using a TSKgel G3000SW XL analytical size-exclusion column (Tosoh) equilibrated in a 25 mMpotassiumphosphate, 125 mM sodium chloride, 200 mM L-ArginineMonohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.

10.3 Binding of Bispecific Monovalent Antibodies Targeting 4-1BB and FAP

10.3.1 Surface Plasmon Resonance (Simultaneous Binding)

The capacity of binding simultaneously human 4-1BB Fc(kih) and human FAPwas assessed by surface plasmon resonance (SPR). All SPR experimentswere performed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany).

Biotinylated human 4-1BB Fc(kih) was directly coupled to a flow cell ofa streptavidin (SA) sensor chip. Immobilization levels up to 1000resonance units (RU) were used. The bispecific antibodies targeting4-1BB and FAP were passed at a concentration range of 250 nM with a flowof 30 μL/minute through the flow cells over 90 seconds and dissociationwas set to zero sec. Human FAP was injected as second analyte with aflow of 30 μL/minute through the flow cells over 90 seconds at aconcentration of 250 nM. The dissociation was monitored for 120 sec.Bulk refractive index differences were corrected for by subtracting theresponse obtained in a reference flow cell, where no protein wasimmobilized. All bispecific constructs could bind simultaneously human4-1BB and human FAP.

10.3.2 Binding on Cells

10.3.2.1 Binding on Human 4-1BB Expressing Cells: Resting and ActivatedHuman Peripheral Mononuclear Blood Leukocytes (PBMC)

Human PBMCs were isolated, used freshly (e.g. resting T cells) oractivated to induce 4-1BB and used to test binding properties ofantibodies to human 4-1BB as already described under Example 7.1.2.

To compare monovalent 4-1BB-binding of 4-1BB FAP-targeted 1+1 constructswith the bivalent-4-1BB-binding of parental anti-4-1BB specific huIgG1P329G LALA clones, constructs were incubated with resting or activated,human 4-1BB expressing PBMCs and detected with PE-conjugated human IgGFc-specific goat IgG F(ab′) as described earlier. As shown in FIGS. 39Dand 39F, conversion into monovalent-4-1BB FAP-targeted 1+1 formatsincreased the EC₅₀ values of 4-1BB binding (Table 67) and decreased theMFI value (FIG. 39B/E and 39C/F) compared to their parental huIgG1 P329GLALA constructs. This increase of EC₅₀ and decrease of MFI lead to amore or less bisection of the area under the curve (AUC) of bindingcurves as shown in FIG. 40.

TABLE 67 EC₅₀ values of binding to activated human CD8⁺ T cells CloneEC₅₀ [nM] 25G7 11.1 25G7/FAP 28H1 1 + 1 >70 25G7/DP47 1 + 1 >70 12B3 0.412B3/FAP 28H1 1 + 1 810.3.2.2 Binding to Human FAP-expressing Cells

The binding to FAP-expressing tumor cell has already been describedabove under 9.2.2.2.

As shown in FIG. 41, the FAP-targeted 1+1 molecules, but not theDP47-targeted 1+1 or parental huIgG1 P293G LALA antibodies of clones25G7 and 12B3 bind efficiently to human FAP-expressing cells (FIGS. 41B/D and 41E/F). Therefore also the 1+1 constructs can specifically bindto FAP-expressing cells, though due to the monovalent FAP-targeting witha slightly higher EC₅₀ than the FAP-targeted 2+2 formats (Table 68 andFIGS. 41 B/D and 41E/F). This however has only minimal effect on the AUCof the binding curves (FIG. 42).

TABLE 68 EC₅₀ values of binding to FAP expressing cell lineNIH/3T3-huFAP clone 19 and WM-266-4 EC₅₀ [nM] with NIH/3T3-huFAP cloneEC₅₀ [nM] with WM- Clone 19 cells 266-4 cells 12B3/FAP 28H1 1 + 1 2.41.7 25G7/FAP 28H1 1 + 1 4.4 5 25G7/DP47 1 + 1 n.d. n.d.10.3.3. NFκB Activation

The protocol to analyze functional capacity using the human 4-1BBexpressing reporter cell line HeLa-hu4-1BB-NFκB-luc has already beendescribed in Example 9.2.3.2.

FAP (28H1)-targeted clone 25G7 or 12B3 1+1 constructs triggeredactivation of the NFκB signaling pathway in the reporter cell line inthe presence of NIH/3T3-huFAP cells (FIGS. 43F and 43I). In contrast,the untargeted control molecule (4-1BB (25G7)×DP47 1+1) or the parentalhuIgG1 P329G LALA antibodies failed to trigger such an effect at any ofthe tested concentrations. This activity of FAP-targeted anti-human4-1BB1+1 antibodies was strictly dependent on a high expression of FAP at thecell surface of added FAP+ cells as no NF-κB activation could bedetected in the absence of FAP-expressing tumor cells (FIGS. 43D and43G) or in the presence of a tumor cell line expressing less FAP(WM-266-4) (FIGS. 43E and 43H). Comparing FAP-targeted 1+1 with theFAP-targeted 2+2 molecules clone 12B3 and 25G7 are behaving differently.The 12B3 FAP-targeted 1+1 construct has a higher EC50 but also higheractivation plateau than the 12B3 FAP-targeted 2+2 construct (FIG. 43F)and therefore both constructs have a similar AUC (FIG. 44A), whereas the25G7 FAP-targeted 1+1 construct has a higher EC₅₀ value and the sameactivation plateau as the 25G7 FAP-targeted 2+2 construct (FIG. 44I) andtherefore a smaller AUC (FIG. 44A) e.g. the 25G7 FAP-targeted 1+1 isclearly performing worse than the 25G7 FAP-targeted 2+2. Reasons couldbe the difference between strong (e.g. 12B3) and low (e.g. 25G7) 4-1BBbinder which may become more pronound with a monovalent 4-1BB-binding.

TABLE 69 EC₅₀ values of activation of the NFκB signaling pathway in thepresence of FAP-expressing tumor cells EC₅₀ [nM] with NIH/3T3- ClonehuFAP clone 19 12B3/FAP 28H1 1 + 1 0.9 25G7/FAP 28H1 1 + 1 ~2

Example 11 Preparation, Purification and Characterization of BispecificAntibodies with a Bivalent Binding to 4-1BB and a Monovalent Binding toTumor Associated Antigen (TAA)

11.1. Generation of Bispecific Antibodies with a Bivalent Binding to4-1BB and a Monovalent Binding to Tumor Associated Antigen (TAA) (2+1Format)

Bispecific agonistic 4-1BB antibodies with bivalent binding for 4-1BBand monovalent binding for FAP, also termed 2+1, have been prepared asdepicted in FIG. 36.

In this example, the first heavy chain HC1 of the construct wascomprised of the following components: VHCH1 of anti-4-1BB binder,followed by Fc knob, at which C-terminus a VL or VH of anti-FAP binderwas fused. The second heavy chain HC2 was comprised of VHCH1 ofanti-4-1BB followed by Fc hole, at which C-terminus a VH or VL,respectively, of anti-FAP binder (clone 4B9) was fused. The generationand preparation of FAP binder 4B9 is described in WO 2012/020006 A2,which is incorporated herein by reference. Binders against 4-1BB(12B3,9B11, 11D5 and 25G7), were generated as described in Example 6.Combination of the targeted anti-FAP-Fc knob with the anti-4-1BB-Fc holechain allows generation of a heterodimer, which includes a FAP bindingmoiety and two 4-1BB binding Fabs (FIGS. 36A and 36B).

The Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced inthe constant region of the knob and hole heavy chains to abrogatebinding to Fcgamma receptors according to the method described inInternational Patent Appl. Publ. No.WO2012/130831A1.

The bispecific 2+1 anti-4-1BB anti-FAP huIgG1 P329GLALA antibodies wereproduced by co-transfecting HEK293-EBNA cells with the mammalianexpression vectors using polyethylenimine. The cells were transfectedwith the corresponding expression vectors in a 1:1:1 ratio (“vector knobheavy chain”:“vector light chain”:“vector hole heavy chain”). Theconstructs were produced and purified as described for the bispecificbivalent anti-4-1BB and anti-FAP huIgG1 P329GLALA antibodies (seeExample 9.1).

The base pair and amino acid sequences for 2+1 anti-4-1BB, anti-FAPconstructs with a-FAP VH fused to knob and VL fused to hole chain can befound respectively in Table 70.

TABLE 70 cDNA and amino acid sequences of mature bispecific 2 + 1anti-4-1BB, anti-FAP human IgG1 P329GLALA. (Constructs with a-FAP VLfused to hole and VH fused to knob chain, termed in Table 72 belowhole-VL) SEQ ID NO: Description Sequence 259 (12B3) VLCL-lightGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC chainATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCA (nucleotide sequence)GTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATCATTCGTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 316 (12B3) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VH (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence ofGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC HC 1)GACAGGCCCCTGGACAAGGGCTGGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGC 317 (12B3) VHCH1 Fc holeCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VL (4B9) (nucleotideAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC sequence of HC2)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTGGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 261 (12B3) VLCL-lightDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP chainGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 318 (12B3) VHCH1Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knob VH (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 1)YMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVS S 319 (12B3) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VL (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 263(25G7) VLCL-light TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGC chainCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGAC (nucleotide sequence)AGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCTTGATAGGCGCGGTATGTGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCA GC 320 (25G7) VHCH1 FcGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGC knob VH (4B9)AGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGC (nucleotide sequence,GGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCG heavy chain 1)CCAGGCCCCTGGAAAAGGCCTGGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACCTACTACGCCGATTCTGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGCGCCAGGGACGACCCCTGGCCCCCCTTTGATTATTGGGGACAGGGCACCCTCGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGG GGACAGGGCACCCTGGTCACCGTGTCCAGC321 (25G7) VHCH1 Fc hole GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGC VL (4B9)(nucleotide AGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGC sequence, heavy chainGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGCG 2)CCAGGCCCCTGGAAAAGGCCTGGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAGCACCTACTACGCCGATTCTGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGCGCCAGGGACGACCCCTGGCCCCCCTTTGATTATTGGGGACAGGGCACCCTCGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG 265 (25G7) VLCL-lightSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKP chainGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSLDRRGMWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS 322 (25G7)VHCH1 Fc EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ knob VH (4B9) (heavyAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL chain 1)YLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 323 (25G7) VHCH1 Fc holeEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ VL (4B9)APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL (heavy chain 2)YLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 267(11D5) VLCL-light GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC chainATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCA (nucleotide sequence)GTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGCTTAATTCGTATCCTCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 324 (11D5) VHCH1 FcCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGA knob VH (4B9)AACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCTTC (nucleotide sequence,CGGCGGCACCTTCAGCAGCTACGCCATTTCTTGGGTGC heavy chain 1)GCCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAGCACCCTGATCTACGGCTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCTAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGC 325 (11D5) VHCH1 Fc holeCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGA VL (4B9)AACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCTTC (nucleotide sequence,CGGCGGCACCTTCAGCAGCTACGCCATTTCTTGGGTGC heavy chain 2)GCCAGGCCCCTGGACAGGGCCTGGAATGGATGGGCGGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAGCACCCTGATCTACGGCTACTTCGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCTAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 269 (11D5) VLCL-lightDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP chainGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLNSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 326 (11D5) VHCH1Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knob VH (4B9) (heavyAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA chain 1)YMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 327 (11D5) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VL (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 271(9B11) VLCL-light GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGC chainATCTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCA (nucleotide sequence)GTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGTTAATTCTTATCCGCAGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 328 (9B11) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VH (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 1)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGC 329 (9B11) VHCH1 Fc holeCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VL (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 2)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 273 (9B11) VLCL-lightDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP chainGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQVNSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 330 (9B11) VHCH1Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knob VH (4B9) (heavyAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA chain 1)YMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVS S 331 (9B11) VHCH1 Fc holeQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VL (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK

The base pair and amino acid sequences for 2+1 anti-4-1BB, anti-FAPconstructs with a-FAP VL fused to knob and VH fused to hole chain can befound respectively in Table 71.

TABLE 71 cDNA and amino acid sequences of mature bispecific 2 + 1anti-4-1BB, anti-FAP human IgG1 P329GLALA. (constructs with a-FAP VHfused to hole and VL fused to knob chain, termed below hole-VH) SEQ IDNO: Description Sequence 259 (12B3) VLCL-light see Table 70 chain(nucleotide sequence) 332 (12B3) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VL (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence ofGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC HC 1)GACAGGCCCCTGGACAAGGGCTGGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 333 (12B3) VHCH1 Fc holeCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VH (4B9) (nucleotideAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC sequence of HC2)GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTGGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTGAATTCCGTTTCTACGCTGACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGC 261 (12B3) VLCL-light see Table 70chain 334 (12B3) VHCH1 Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knobVL (4B9) APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 1)YMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQGIMLPPTFGQGTKVEIK335 (12B3) VHCH1 Fc hole QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VH(4B9) APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSEFRFYADFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 263 (25G7) VLCL-light see Table 70chain (nucleotide sequence) 336 (25G7) VHCH1 FcGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC knob VL (4B9)AGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC (nucleotide sequence,GGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCG heavy chain 1)CCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG 337 (25G7) VHCH1 Fc holeGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTAC VH (4B9) (nucleotideAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCC sequence, heavy chainGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCG 2)CCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGACGACCCGTGGCCGCCGTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGAC AGGGCACCCTGGTCACCGTGTCCAGC 265(25G7) VLCL-light see Table 70 chain 338 (25G7) VHCH1 FcEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ knob VL (4B9) (heavyAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL chain 1)YLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 339(25G7) VHCH1 Fc hole EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ VH (4B9)APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTL (heavy chain 2)YLQMNSLRAEDTAVYYCARDDPWPPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 267 (11D5) VLCL-light see Table 70chain (nucleotide sequence) 340 (11D5) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VL (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 1)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 341 (11D5) VHCH1 Fc holeCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VH (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 2)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTACTCTGATCTACGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGG GGACAGGGCACCCTGGTCACCGTGTCCAGC269 (11D5) VLCL-light see Table 70 chain 342 (11D5) VHCH1 FcQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knob VL (4B9) (heavyAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA chain 1)YMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 343(11D5) VHCH1 Fc hole QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VH (4B9)APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSTLIYGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS 271 (9B11) VLCL-light see Table 70chain (nucleotide sequence) 344 (9B11) VHCH1 FcCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA knob VL (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 1)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCTGGGGGCGGAGGTTCCGGAGGCGGTGGATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTCCCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGGGCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCAAGGTGGAAATC AAG 345 (9B11) VHCH1 Fc holeCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGA VH (4B9)AGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCC (nucleotide sequence,GGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGC heavy chain 2)GACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGATCTTCTGGTGCTTACCCGGGTTACTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCGGCGGCGGAGGTTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATCATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGCGGCTTCAACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGC 273 (9B11) VLCL-light see Table 70chain 346 (9B11) VHCH1 Fc QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ knobVL (4B9) (heavy APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA chain 1)YMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQGIMLPPTFGQGTKVEIK347 (9B11) VHCH1 Fc hole QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ VH(4B9) APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTA (heavy chain 2)YMELSSLRSEDTAVYYCARSSGAYPGYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSS

TABLE 72 Biochemical analysis of bispecific constructs with a bivalentbinding to 4-1BB and a monovalent binding to FAP (2 + 1 4-1BB/FAP humanIgG1 P329GLALA) Yield Monomer CE-SDS Clone [mg/l] [%] (nonred) 2 + 125G7/FAP (hole-VH) 25.6 96.7 95.4 2 + 1 11D5/FAP (hole-VH) 6.3 97 89.211.2. Binding of Bispecific Monovalent Antibodies Targeting 4-1BB andFAP11.2.1. Surface Plasmon Resonance (Simultaneous Binding)

The capacity of binding simultaneously to human 4-1BB Fc(kih) and humanFAP was assessed by surface plasmon resonance (SPR). All SPR experimentswere performed on a Biacore T200 at 25° C. with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,Biacore, Freiburg/Germany).

Biotinylated human 4-1BB Fc(kih) was directly coupled to a flow cell ofa streptavidin (SA) sensor chip. Immobilization levels up to 400resonance units (RU) were used. The bispecific antibodies targeting4-1BB and FAP were passed at a concentration range of 200 nM with a flowof 30 μL/minute through the flow cells over 90 seconds and dissociationwas set to zero sec. Human FAP was injected as second analyte with aflow of 30 μL/minute through the flow cells over 90 seconds at aconcentration of 500 nM. The dissociation was monitored for 120 sec.Bulk refractive index differences were corrected for by subtracting theresponse obtained in a reference flow cell, where no protein wasimmobilized.

As shown in FIGS. 37B and 37C, all bispecific constructs could bindsimultaneously to human 4-1BB and human FAP.

11.2.2 NFκB Activation

The generation of NFκB-reporter cell line HeLa-huCD137-NFκB-luc clone 26as well as the set up of the activation assay has already been describedunder 9.2.3.

The tested FAP (4B9)-targeted 2+1 constructs containing clone 11D5 (FIG.44A-44C) or clone 25G7 (Figure G-I) triggered activation of the NFkBsignaling pathway in the reporter cell line in the presence ofFAP-expressing tumor cells. This activity was strictly dependent on theexpression of FAP at the cell surface of tumor cells as no NF-κBactivation could be detected in the absence of FAP-expressing tumorcells (FIGS. 43D and 43G). Different to the other tested formats (e.g.2+2, 1+1) the FAP (4B9)-targeted 2+1 could also induce an activation inthe presence of lower FAP-expressing WM-266-4 cells (FIGS. 44E and H).In the presences of NIH/3T3-huFAP clone 19 binders the 2+1 fromats arealso superior compared to the FAP (28H1)-targeted 2+2 or 1+1 constructs(FIGS. 43F and 43I, FIG. 44B). This may be explained with the strongerFAP-binder (4B9>28H1) and the different ratio between FAP-binding sidesand 4-1BB binding sides (1:2 versus 1:1).

TABLE 73 EC₅₀ values of activation of the NFκB signaling pathway in thepresence of FAP-expressing tumor cells EC₅₀ [nM] with NIH/3T3-huFAP EC₅₀[nM] with Clone clone 19 cells WM-266-4 4-1BB(25G7)/ 0.1 0.3 FAP(4B9)2 + 1 4-1BB(11D5)/ 0.3 0.4 FAP(4B9) 2 + 1

What is claimed is:
 1. An antibody or antibody fragment thereof thatspecifically binds to OX40, wherein said antibody or fragment thereofcomprises: (a) a heavy chain variable domain (VH) comprising (i) anCDR-H1 comprising the amino acid sequence of SEQ ID NO:2; (ii) an CDR-H2comprising the amino acid sequence of SEQ ID NO:4; (iii) an CDR-H3comprising the amino acid sequence of SEQ ID NO:7; and (b) a light chainvariable domain (VL) comprising (i) an CDR-L1 comprising the amino acidsequence of SEQ ID NO:13; (ii) an CDR-L2 comprising the amino acidsequence of SEQ ID NO:16; and (iii) an CDR-L3 comprising the amino acidsequence of SEQ ID NO:20.
 2. The antibody or fragment thereof of claim1, wherein the VH has at least 95% amino acid sequence identity to SEQID NO:27, and the VL has at least 95% amino acid sequence identity toSEQ ID NO:28.
 3. The antibody or fragment thereof of claim 1, whereinthe VH has at least 97% amino acid sequence identity to SEQ ID NO:27,and the VL has at least 97% amino acid sequence identity to SEQ IDNO:28.
 4. The antibody or fragment thereof of claim 1, wherein the VHhas at least 99% amino acid sequence identity to SEQ ID NO:27, and theVL has at least 99% amino acid sequence identity to SEQ ID NO:28.
 5. Theantibody or fragment thereof of claim 1, wherein the VH comprises theamino acid sequence of SEQ ID NO:27, and the VL comprises the amino acidsequence of SEQ ID NO:28.
 6. The antibody or fragment thereof of claim 1that is a monoclonal antibody.
 7. The antibody or fragment thereof ofclaim 5 that is a monoclonal antibody.
 8. The antibody or fragmentthereof of claim 6 that is a humanized or human antibody.
 9. Theantibody or fragment thereof of claim 7 that is a humanized or humanantibody.
 10. The antibody or fragment thereof of claim 1 that is ascFv, a Fab, or a Fab' fragment.
 11. The antibody or fragment thereof ofclaim 5 that is a scFv, a Fab, or a Fab' fragment.
 12. The antibody orfragment thereof of claim 1 that is an antibody comprising an Fc domainof the human IgG1 subclass.
 13. The antibody of claim 12 that comprisesthe amino acid mutations L234A, L235A and P329G (numbering according toKabat EU index).
 14. The antibody or fragment thereof of claim 5 that isan antibody comprising an Fc domain of the human IgG1 subclass.
 15. Theantibody of claim 14 that comprises the amino acid mutations L234A,L235A and P329G (numbering according to Kabat EU index).
 16. An antibodythat specifically binds to OX40, comprising: (a) a light chaincomprising the amino acid sequence of SEQ ID NO:186; and (b) a heavychain comprising the amino acid sequence of SEQ ID NO:187.
 17. Theantibody of claim 16 that is a monoclonal antibody.
 18. A compositioncomprising the antibody or fragment of claim 1 and a pharmaceuticallyacceptable excipient.
 19. A composition comprising the antibody orfragment of claim 5 and a pharmaceutically acceptable excipient.
 20. Acomposition comprising the antibody or fragment of claim 16 and apharmaceutically acceptable excipient.